Note pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Absolute Configuration of the Cytotoxic Marine Alkaloid Monanchocidin A Larisa K. Shubina, Tatyana N. Makarieva,* Alla G. Guzii, Vladimir A. Denisenko, Roman S. Popov, Pavel S. Dmitrenok, and Valentin A. Stonik G. B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Prospect 100-let Vladivostoku 159, Vladivostok 690022, Russian Federation S Supporting Information *
ABSTRACT: The absolute configuration of the cytotoxic guanidine alkaloid monanchocidin A with 11 stereogenic centers from the marine sponge Monanchora pulchra was determined as 5R, 8S, 10S, 13R, 14S, 15R, 19R, 23R, 37S, 42S, 43R after extensive reductive degradation and conversion of the resulting alcohols to MTPA derivatives.
P
attempts at chemical transformations such as oxidation, hydrogenation, metal hydride reduction, and acid or alkaline hydrolysis were unsuccessful with ptilomycalin A and its derivatives and resulted only in intractable mixtures of products. Here, we describe the determination of the absolute configurations of all of the stereogenic centers in 1 using reductive degradation and the modified Mosher’s method. Previously, the relative configurations of the PG core of monanchocidin A, containing seven stereogenic centers, and the morpholinone part, containing three stereogenic centers, were deduced from ROESY experiments8 (Figure S1). The relative configuration of C-23 was not assigned. Moreover, no absolute configurations of any of the stereocenters in 1 have been established. Earlier we obtained triol 2 by reductive cleavage of hemiaminal and hemiacetal groups during the treatment of 1 with sodium borohydride at 65 °C in EtOH.8 It might have been used for the determination of absolute configurations by Mosher’s method,10 but the yield of this product was too small. After re-collection of the sponge, we were able to obtain an additional amount of triol 2 using the same method8 with slight modifications. The obtained triol 2 was converted into the tetra-(S)-MTPA (2a) and tetra-(R)-MTPA (2b) derivatives by acylation of the three hydroxy groups and the amino group with (R)- and (S)-α-methoxy-α-(trifluoromethyl)phenyl acetyl chloride (MTPACl), respectively, by using dimethylaminopyridine (DMAP) as catalyst (Scheme 1). The tetra-MTPA derivatives were isolated and purified using column chromatography on YMC gel ODS A. Interpretation of the 1H NMR chemical shift differences ΔδSR (δS − δR) between 2a and 2b indicated the 5R, 19R, 37S absolute configurations in 1. Taking into account the relative configuration of 1 (Figure S1), the absolute configuration of
entacyclic guanidine alkaloids (PGAs) are the most complicated guanidine-containing marine alkaloids isolated to date.1,2 The structural novelty and a wide range of biological activities demonstrated by these molecules have attracted significant attention.1−3 Ptilomycalin A, the first representative of PGAs, was discovered in 1989.4 The relative configuration of its pentacyclic core with seven stereogenic centers and a sevenmembered spiro-ring was determined by NOESY and ROESY experiments.4,5 The first determination of the absolute configuration of the pentacyclic core of alkaloids belonging to this structural group was carried out in 1993 after ozonolysis of crambescidin 816, closely related to ptilomycalin A.6 Later, an enantioselective total synthesis of (−)-ptilomycalin A rigorously established the absolute configuration of ptilomycalin A and other known PGAs, containing a seven-membered spiro-ring residue.7 Monanchocidin A (1), containing a five-membered spiro-ring in the PG core along with an unusual branched long alkyl chain and a heavily oxygenated morpholinone fragment, was first isolated from the Far Eastern marine sponge Monanchora pulchra in 2010.8 This compound, cytotoxic against tumor cells,9 contains 11 stereogenic centers and remains a challenge for absolute configuration assignment because its molecule does not contain chromophores, secondary hydroxy groups, or a double bond in the spiro-ring of the PG core, in contrast with ptilomycalins and crambescidins. Therefore, it is not possible to apply an electronic circular dichroism (ECD) method, Mosher’s method,10 or ozonolysis. Moreover, monanchocidin A is a noncrystalline compound. This type of natural compound with a high H/C ratio is not suitable for the determination of configurations by X-ray analysis (so-called “Crew’s rule”11). Another difficulty is the fact that compounds with many potential sites for reactivity like monanchocidin A are poor candidates for directed chemical transformations. For example, Kashman,4 who first discovered ptilomycalin A, a simpler compound compared to monanchocidin A, noted that initial © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 2, 2018
A
DOI: 10.1021/acs.jnatprod.8b00105 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
using a Shimadzu Instrument equipped with a RID-10A refractive index detector and YMC-ODS-A (250 × 10 mm) column. Animal Material. The sponge Monanchora pulchra (Lambe, 1894) was collected by dredging during the 43th scientific cruise of R/V Academic Oparin in August 2012, near Urup Island (45°53,6 N; 149°38,6 E; depth 137 m) and identified by V. B. Krasokhin. A voucher specimen is kept under the registration number PIBOC O43603 in the marine invertebrate collection of the Pacific Institute of Bioorganic Chemistry (Vladivostok, Russia). Extraction and Isolation. A sample of M. pulchra (dry weight 10 g) was extracted with EtOH (200 mL × 2). The extract was concentrated under reduced pressure and subjected to YMC gel ODS A flash chromatography, using H2O, EtOH−H2O (40:60, v/v), and EtOH−H2O (60:40, v/v + 0.05% TFA) eluent systems. The fraction eluted with 60% EtOH with 0.05% tetrafluoroacetic acid (TFA) was purified by reversed-phase HPLC using EtOH−H2O (65:35, v/v + 0.05% TFA) to afford monanchocidin A (10 mg, 0.001% from dry weight), which was structurally identified by comparison of its NMR spectra, ESIMS, and [α]D data with an authentic sample. Compound 2. To a solution of 1 (6 mg) in EtOH (0.5 mL) was added NaBH4 (1.5 mg, excess). The mixture was stirred at 65 °C for 10 h. After the reaction was finished, the solution was neutralized with TFA and the solvent was removed by evaporation. The residue was purified by HPLC using EtOH−H2O (65:35 + 0.05% TFA) to afford 2 (3 mg, 56% yield) as a colorless oil: [α]D −7 (c 0.4, EtOH); IR (CHCl3) νmax 2928, 1724, 1675, 1636 cm−1; 1H NMR (700 MHz, CD3OD) δ 5.71 (1H, dt, J = 6.5, 15.5 Hz, H-3), 5.44 (1H, ddt, J = 1.6, 7.0, 15.5 Hz, H-4), 4.90 (1H, quint, J = 6.3 Hz, H-23), 4.02 (1H, m, H5), 4.01 (1H, dd, J = 3.9, 8.0 Hz, H-37), 3.87 (1H, dt, J = 7.0, 10.6 Hz, H-13), 3.79 (1H, m, H-10), 3.72 (1H, m, H-19), 3.63 (1H, m, H-15), 3.48 (1H, m, H-8), 3.32 (2H, m, H-39), 2.93 (2H, brt, J = 7.1, H-41), 2.43 (1H, m, H-14), 2.29 (1H, m, H-9a), 2.26 (1H, m, H-6a), 2.24 (1H, m, H-11a), 2.18 (1H, m, H-12a), 2.06 (2H, quint, J = 7.0 Hz, H2), 1.85 (2H, quint, J = 7.0 Hz, H-40), 1.81 (1H, m, H-12b), 1.79 (1H, m, H-36a), 1.74 (2H, m, H-17), 1.65 (1H, m, H-7a), 1.62 (2H, m, H42), 1.60 (1H, m, H-24a), 1.58 (2H, m, H-6b, H-36b), 1.57 (1H, m, H-24b), 1.56 (2H, m, H-16), 1.44 (2H, m, H-18), 1.31 (2H, m, H-9b), 1.30 (1H, m, H-7b), 1.27−1.36 (22 H, quint, H-25−H-35), 1.16 (3H, d, J = 6.3 Hz, H-20), 1.00 (3H, t, J = 7.3 Hz, H-1), 0.90 (3H, t, J = 7.3 Hz, H-43); 13C NMR (CD3OD, 125 MHz) δ 179.2 (C-38), 171.8 (C22), 151.0 (C-21), 135.1 (C-3), 133.5 (C-4), 79.4 (C-23), 73.7 (C37), 73.4 (C-5), 68.7 (C-19), 60.0 (C-13), 58.4 (C-10), 54.3 (C-15), 52.2 (C-8), 51.3 (C-14), 38.8 (C-41), 37.0 (C-39), 36.4 (C-17), 35.1 (C-9), 35.0 (C-16), 34.4 (C-36), 31.3 (C-6), 31.2 (C-11), 31.2−31.5 (C-25−C-35), 30.2 (C-12), 29.4 (C-40), 28.5 (C-7), 28.5 (C-24), 28.3 (C-46), 27.2 (C-18), 26.7 (C-2), 24.2 (C-20), 14.5 (C-1), 10.7 (C47); HRESIMS m/z 762.6101 [M + H]+ (calcd for C43H80N5O6, 762.6103). Preparation of MTPA Derivatives of 2. The sample of 2 (1 mg each) was treated with (R)- or (S)-MTPACl (10 μL) and DMAP in dry pyridine (100 μL) in a 4 mL sealed vial. After stirring for 30 min at rt, the reaction mixture was concentrated under reduced pressure. The residue was then applied directly onto a reversed-phase (YMC gel ODS A) column successively eluting with 60% → 100% EtOH−H2O (gradient of EtOH increased by 10%) to afford five fractions. All the fractions were monitored by MS. Fractions that eluted with 80% EtOH afforded the tetra-(S)- or (R)-MTPA derivatives (2a/2b). (S)-MTPA derivative (2a): colorless oil; 1H NMR (700 MHz, CD3OD) δ 5.87 (1H, m, H-3), 5.44 (1H, m, H-5), 5.38 (1H, m, H-4), 5.11 (1H, m, H-19), 5.02 (1H, m, H-37), 3.32 (2H, m, H-39), 3.30 (2H, m, H-41), 2.06 (2H, m, H-2), 1.86 (1H, m, H-6a), 1.82 (1H, m, H-36a), 1.78 (1H, m, H-36b), 1.76 (1H, m, H-6b), 1.71 (2H, m, H40), 1.62 (1H, m, H-18a), 1.54 (1H, m, H-18b), 1.35 (3H, d, J = 6.0 Hz, H-20), 0.97 (3H, t, J = 7.3 Hz, H-1); HRESIMS m/z 1626.7693 [M + H]+ (calcd for C83H108 F12N5O14, 1626.7696). (R)-MTPA derivative (2b): colorless oil; 1H NMR (700 MHz, CD3OD) δ 5.98 (1H, m, H-3), 5.51 (1H, m, H-5), 5.49 (1H, m, H-4), 5.13 (1H, m, H-19), 5.08 (1H, m, H-37), 3.28 (2H, m, H-39), 3.21 (2H, m, H-41), 2.10 (2H, m, H-2), 1.88 (1H, m, H-36a), 1.83 (2H, m, H-6a, H-36b), 1.74 (1H, m, H-6b), 1.71 (1H, m, H-18a), 1.69 (2H, m,
Scheme 1. (A) Preparation of Triol 2 and Its MTPA Derivatives (2a, 2b) and the ΔδSR (δS − δR) Values of 2a,b; (B) Preparation of Diol 3 and Its MTPA Derivatives (3a, 3b) and the ΔδSR (δS − δR) Values of 3a,b
the vessel part of monanchocidin was proved to be 5R, 8S, 10S, 13R, 14S, 15R, 19R, while the configuration of the morpholinone part was established as 37S, 42S, 43R. To determine the configuration at C-23, it was necessary to break the C-22 ester bond. Acid or alkaline hydrolysis failed. Only after stronger reductive degradation of 1 by the treatment of 1 with NaBH4 in EtOH at 78 °C did we succeed in obtaining diol 3. The absolute configuration at C-23 was then assigned by application of the modified Mosher’s method.10 Derivatization of 3 with (R)- and (S)-MTPACl yielded the tri-(S)-MTPA derivative 3a and tri-(R)-MTPA derivative 3b, respectively. The observed chemical shift differences ΔδSR (δS − δR) between 3a and 3b (Scheme 1) indicated the 23R configuration, and therefore the absolute configuration of the whole molecule of monanchocidin A was assigned as 5R, 8S, 10S, 13R, 14S, 15R, 19R, 23R, 37S, 42S, 43R (Scheme 1). These configurations are the mirror image of the previously arbitrarily shown PG core structure of the monanchocidins, monanchomycalins, normonanchocidins, and monanchoxymycalin A, which contain a fivemembered spiro-ring,12−15 but the same as the morpholinone residue in monanchocidins A, D, and E.12 Thus, the absolute configuration of monanchocidin A (1) was determined by reductive degradation of 1 into triol 2 and diol 3, followed by application of the modified Mosher’s method. The determination of the absolute configurations of all of the stereogenic centers in 1 should be useful to carry out stereoselective syntheses of this lead structure and/or analogues for further drug development.
■
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured using a JASCO DIP-360 polarimeter. IR spectra were recorded using a Bruker Vector 22 spectrophotometer. The 1H and 13 C NMR spectra were obtained using a Bruker Avance III 700 spectrometer. Chemical shifts were referenced to the corresponding residual solvent signal (δH 3.30/δC 49.60 for CD3OD). ESI mass spectra (including HRESIMS) were measured using a Bruker Impact II Q-TOF mass spectrometer (Bruker Daltonics). HPLC was performed B
DOI: 10.1021/acs.jnatprod.8b00105 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
H-40), 1.61 (1H, m, H-18b), 1.28 (3H, d, J = 6.0 Hz, H-20), 1.01 (3H, t, J = 7.3 Hz, H-1); HRESIMS m/z 1626.7693 [M + H]+ (calcd for C83H108 F12N5O14, 1626.7696). Compound 3. To a solution of 1 (4 mg) in EtOH (0.5 mL) was added NaBH4 (1.5 mg, excess). The mixture was stirred at 78 °C for 4 h. After the reaction was finished, the solution was neutralized with TFA and the solvent was removed by evaporation. The residue was purified by HPLC using EtOH−H2O (65:35 + 0.05% TFA) to afford 3 (0.8 mg, 40% yield) as a colorless oil: 1H NMR (700 MHz, CD3OD) δ 4.0 (1H, dd, J = 3.9, 8.0 Hz, H-37), 3.42 (1H, m, H-23), 3.32 (2H, m, H-39), 2.93 (2H, brt, J = 6.8, H-41), 1.85 (2H, quint, J = 6.8 Hz, H40), 1.76 (1H, m, H-36a), 1.59 (1H, m, H-36b), 1.47 (2H, m, H-46a, H-24a), 1.45 (1H, m, H-24b), 1.39 (1H, m, H-46b), 1.27−1.36 (22 H, brs, H-25−H-35), 0.92 (3H, t, J = 7.3 Hz, H-47); HRESIMS m/z 371.3278 [M − H]− (calcd for C21H43N2O3, 371.3279). Preparation of MTPA Derivatives of 3. The sample of 3 (0.4 mg each) was treated with (R)- or (S)-MTPACl (10 μL) and DMAP in dry pyridine (100 μL) in a 4 mL sealed vial. After stirring for 30 min at rt, the reaction mixture was concentrated under reduced pressure. The residue was then applied directly onto a reversed-phase (YMS gel ODS A) column successively eluting with 60% → 100% EtOH−H2O (gradient of EtOH increased by 10%) to afford five fractions. All the fractions were monitored by MS. Fractions that eluted with 90% EtOH afforded the tri-(S)- or (R)-MTPA derivatives (3a/3b). (S)-MTPA derivative (3a): colorless oil; 1H NMR (700 MHz, CD3OD) δ 5.02 (1H, m, H-37), 5.015 (1H, m, H-23), 3.30 (4H, m, H-39, H-41), 1.83 (1H, m, H-36a), 1.78 (1H, m, H-36b), 1.76 (2H, m, H-40), 1.68 (1H, m, H-46a), 1.66 (1H, m, H-46b), 1.56 (1H, m, H24a), 1.54 (1H, m, H-24b), 0.92 (3H, t, J = 7.3 Hz, H-47); HRESIMS m/z 1043.4460 [M + Na]+ (calcd for C51H65 F9N2O9Na, 1043.4439). (R)-MTPA derivative (3b): colorless oil; 1H NMR (700 MHz, CD3OD) δ 5.07 (1H, m, H-37), 5.02 (1H, m, H-23), 3.26 (4H, m, H39, H-41), 1.86 (1H, m, H-36a), 1.84 (1H, m, H-36b), 1.72 (2H, m, H-40), 1.64 (1H, m, H-24a), 1.61 (1H, m, H-46a), 1.59 (1H, m, H46b), 1.58 (1H, m, H-24b), 0.80 (3H, t, J = 7.3 Hz, H-47); HRESIMS m/z 1043.4460 [M + Na]+ (calcd for C51H65 F9N2O9Na, 1043.4439).
■
(2) Berlinck, R. G. S.; Romminger, S. Nat. Prod. Rep. 2016, 33, 456− 490 and other reviews in this series.. (3) Shi, Y. L.; Moazami, Y.; Pierce, J. G. Bioorg. Med. Chem. 2017, 25, 2817−2824. (4) Kashman, Y.; Hirsh, S.; McConnell, O. J.; Ohtani, I.; Kusumi, T.; Kakisawa, H. J. Am. Chem. Soc. 1989, 111, 8925−8926. (5) Ohtani, I.; Kusumi, T.; Kakisawa, H.; Kashman, Y.; Hirsh, S. J. Am. Chem. Soc. 1992, 114, 8472−8479. (6) Jares-Erijman, E. A.; Ingrum, A. L.; Carney, J. R.; Kenneth, L.; Rinehart, K. L.; Sakai, R. J. Org. Chem. 1993, 58, 4805−4808. (7) Overman, L. E.; Rabinowitz, M. H.; Renhowe, P. A. J. Am. Chem. Soc. 1995, 117, 2657−2658. (8) Guzii, A. G.; Makarieva, T. N.; Denisenko, V. A.; Dmitrenok, P. S.; Kuzmich, A. S.; Dyshlovoy, S. A.; Krasokhin, V. B.; Stonik, V. A. Org. Lett. 2010, 12, 4292−4295. (9) Dyshlovoy, S. A.; Hauschild, J.; Amann, K.; Tabakmakher, K. M.; Venz, S.; Walther, R.; Guzii, A. G.; Makarieva, T. N.; Shubina, L. K.; Fedorov, S. N.; Stonik, V. A.; Bokemeyer, C.; Balabanov, S.; Honecker, F.; von Amsberg, G. Oncotarget 2015, 6, 17328−17341. (10) Seco, J. M.; Quinoa, E.; Riguera, R. Chem. Rev. 2004, 104, 17− 117. (11) Molinski, T. F.; Morinaka, B. I. Tetrahedron 2012, 68, 9307− 9343. (12) Makarieva, T. N.; Tabakmakher, K. M.; Guzii, A. G.; Denisenko, V. A.; Dmitrenok, P. S.; Shubina, L. K.; Kuzmich, A. S.; Lee, H. S.; Stonik, V. A. J. Nat. Prod. 2011, 74, 1952−1958. (13) Makarieva, T. N.; Tabakmakher, K. M.; Guzii, A. G.; Denisenko, V. A.; Dmitrenok, P. S.; Kuzmich, A. S.; Lee, H. S.; Stonik, V. A. Tetrahedron Lett. 2012, 53, 4228−4231. (14) Makarieva, T. N.; Tabakmakher, K. M.; Denisenko, V. A.; Guzii, A. G.; Dmitrenok, P. S.; Kuzmich, A. S.; Stonik, V. A. Nat. Prod. Commun. 2015, 10, 913−916. (15) Tabakmakher, K. M.; Makarieva, T. N.; Shubina, L. K.; Denisenko, V. A.; Guzii, A. G.; Popov, R. S.; Kuzmich, A. S.; Lee, H. S.; Lee, Y. J.; Stonik, V. A. Nat. Prod. Commun. 2016, 12, 1817−1820.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00105. Additional information (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Tel: 7 (423) 231-11-68. Fax: 7 (423) 231-40-50. E-mail:
[email protected]. ORCID
Tatyana N. Makarieva: 0000-0002-2446-8543 Valentin A. Stonik: 0000-0002-8213-8411 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was supported by Grant 17-14-01065 from the Russian Science Foundation, and the study was carried out on the equipment of the Collective Facilities Center of The Far Eastern Center for Structural Molecular Research (NMR/MS) of PIBOC FEB RAS.
■
REFERENCES
(1) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2017, 34, 235−294 and other reviews in this series.. C
DOI: 10.1021/acs.jnatprod.8b00105 J. Nat. Prod. XXXX, XXX, XXX−XXX
