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19-[(1′S,4′R)‑4′-Hydroxy-1′-methoxy-2′-oxopentyl]geldanamycin, a Natural Geldanamycin Analogue from Streptomyces hygroscopicus 17997 Shufen Li,† Siyang Ni,† Linzhuan Wu,*,† Li Li,‡ Bingya Jiang,*,† Hongyuan Wang,† Guizhi Sun,† Maoluo Gan,† Jingyan Li,† Weiqing He,† Ling Lin,† Yiguang Wang,† Shuoke Bai,† and Shuyi Si† †
Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *
ABSTRACT: A novel natural geldanamycin analogue was discovered in Streptomyces hygroscopicus 17997. Its 4,5-dihydro form was also identified in the gdmP gene disruption mutant of Streptomyces hygroscopicus 17997. The structures of the two compounds were determined to be 19-[(1′S,4′R)-4′hydroxy-1′-methoxy-2′-oxopentyl]geldanamycin (1) and 19-[(1′S,4′R)-4′hydroxy-1′-methoxy-2′-oxopentyl]-4,5-dihydrogeldanamycin (2), respectively, by extensive spectroscopic data analysis, including 2D NMR, modified Mosher’s method, and electronic circular dichroism. Compared to geldanamycin, 1 and 2 showed increased water solubility and decreased cytotoxicity against HepG2 cells.
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than GDM.17,18 Natural GDM analogue(s) may have the potential for anticancer agent development. As a result of our sustained efforts with novel natural GDM analogues, we discovered 19-[(1′S,4′R)-4′-hydroxy-1′-methoxy2′-oxopentyl]geldanamycin from S. hygroscopicus 17997, a GDM producer isolated from Chinese soil.16,22 This paper describes the isolation and structure elucidation of this novel GDM analogue and the identification of its 4,5-dihydro form from a mutant strain of S. hygroscopicus 17997. An ethyl acetate (EtOAc) extract of S. hygroscopicus 17997 was loaded onto silica gel TLC for analysis. The developed chromatogram was sprayed with 2.0 mol/L NaOH to detect benzoquinone ansamycins,23 and a positive blue band with an Rf value of 0.22 appeared (Figure 1, indicated by 1 at the boldtype arrow), together with GDM (Rf 0.62) and thiazinogeldanamycin (Rf 0.10). The yellow band at Rf 0.22 (not subjected to spraying with NaOH) was eluted with EtOAc for ESI(+)-MS analysis. A principal peak appeared at m/z 713 (1), whose MS2 fragment ions exhibited a typical pattern of GDM (Supporting Information, Figure S3), suggesting that 1 is a GDM analogue. As 1 is different from any known natural GDM analogue or GDM biosynthetic intermediate in molecular mass, it should be a novel natural GDM analogue. From the 50 L fermentation supernatant of S. hygroscopicus 17997, we obtained 17.5 mg of 1 as an amorphous, yellow
eldanamycin (GDM, Chart 1) is a benzoquinone ansamycin produced by Streptomyces hygroscopicus.1−3
Chart 1. Chemical Structures of GDM, 1, and 2
GDM exhibits exceptional potency against cancer cells, but acts only as a lead compound in anticancer drug development because of its severe liver toxicity and poor water solubility.4,5 Hundreds of GDM analogues had been produced by chemical modification,6−8 and among them, 17-allylamino-17-demethoxygeldanamycin and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin entered into clinical trials for solid tumor treating.9−11 A number of novel GDM analogues with increased water solubility and potent cytotoxicity have been reported in the past few years.12−15 Natural GDM analogues such as thiazinogeldanamycin and 19-S-methylgeldanamycin are the secondary metabolites of S. hygroscopicus 17997 and other GDM producers.16−21 Although some natural GDM analogues showed decreased potency against cancer cells, they exhibited much higher water solubility © XXXX American Chemical Society and American Society of Pharmacognosy
Received: January 23, 2013
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Table 1. 13C and 1H NMR Spectroscopic Data for 1 and 2 (in CDCl3) 1 position
Figure 1. Silica gel TLC (GF254) of EtOAc extract of the fermentation supernatant of Streptomyces hygroscopicus 17997 (a) and its gdmP gene disruption mutant (b), with a mobile phase of EtOAc−CH2Cl2−nhexane−methanol, 9:6:6:1.5, v/v. Blue band (after spraying with 2.0 mol/L NaOH) is designated as 1 and 2, which are novel natural GDM analogues.
powder (purity ≥97%, calculated by area % of HPLC at 307 nm). Compound 1 exhibited a UV profile very similar to GDM (Supporting Information, Figure S1), indicating that 1 was indeed a GDM analogue. The gdmP gene (encodes a cytochrome P450 oxidase for C4,5 desaturation in GDM biosynthesis) disruption mutant of S. hygroscopicus 17997 is a 4,5-dihydrogeldanamycin producer.24 We presume that the gdmP gene disruption mutant should produce the 4,5-dihyro form of 1. The following silica gel TLC and MS/MS data confirmed our expection: a yellow compound with m/z 715 (2) was isolated from the fermentation broth of gdmP gene disruption mutant, and the fragment ions of m/z 715 exhibited a typical MS2 pattern of GDM and its close analogues (Supporting Information, Figure S13). Using the same separation procedure leading to 1, we obtained 37 mg of 2 (purity ≥96%, calculated by area % of HPLC at 307 nm) from 120 L of fermentation supernatant of the gdmP gene disruption mutant. Compound 2 exhibited a UV profile very similar to 4,5-dihydrogeldanamycin (Supporting Information, Figure S1), suggesting again that 2 belonged to the 4,5-dihydro form of 1. The molecular formulas of 1 and 2 were established as C35H50N2O12 and C35H52N2O12, respectively, on the basis of high-resolution electrospray ionization mass spectroscopy (HRESIMS, Supporting Information, Figures S4 and S14). Compound 2 is two hydrogens more than 1, as expected. NMR data demonstrated that 1 was an analogue of GDM. Compared with GDM, 1 lost the H-19 [δH 7.29 s] of GDM25 and gained six new carbons with associated oxygen(s) and/or hydrogen(s) assigned to a methoxyl [δH 3.51 (s), δC 59.8], a methyl [δH 1.26 (d, J = 6.6 Hz), δC 22.3], a methylene [δH 3.11 (dd, J = 19.2, 3.0 Hz, H-3′a), 2.85 (dd, J = 19.2, 9.6 Hz, H-3′b), δC 48.9], two methines [δH 5.20 (s, H-1′), δC 80.8 (C-1′), δH 4.32 (dqd, J = 9.6, 6.6, 3.0, H-4′), δC 63.6 (C-4′)], and a carbonyl (δC 207.2). These new carbons, together with their associated oxygen(s) and/or hydrogen(s), constituted a moiety of 4′-hydroxy-1′-methoxy-2′-oxopentyl attached at C-19 of GDM, as indicated by the HMBC spectrum of 1 displaying the following two- or three-bond correlations: H-1′/C-18, C-19, C20, C-2′, and 1′-OCH3; H2-3′/C-2′, C-4′, and C-5′; H3-5′/C-3′ and C-4′. Other structural fragments of 1 were the same as GDM. Therefore, the planar structure of 1 is 19-(4′-hydroxy-1′methoxy-2′-oxopentyl)geldanamycin. The NMR data of 1 were summarized in Table 1.
δC
2
δH, mult. (J in Hz)
δC
1 2 3 4
173.0 138.7 126.0 128.9
6.88, brd (12.0) 6.37, dd (12.0, 11.5)
172.7 135.6 134.2 22.8
5
131.1
5.32, dd (11.5,10.2)
30.0
6 7 8 9 10
75.0 82.2 129.8 133.4 34.8
4.73, t (10.2, 9.0) 5.03, d (9.0)
11 12
72.2 79.8
13
30.9
14 15
29.0 29.9
5.21, d (12.0) 2.32, ddq (12.0, 10.2, 6.6) 3.62, dd (10.2, 3.0) 2.91, ddd (14.4, 3.0, 3.0) 1.62, ddd (14.4, 10.8, 4.2) 0.73, m 2.17, m 2.65, dd (13.2, 4.8)
78.4 83.4 130.2 132.3 34.5
129.7 156.5 181.6 119.4 143.9 182.8 80.8 207.2 48.9
5.20, s 3.11, dd (19.2, 3.0)
5′ 2-CH3 6-OCH3 7-OCONH2 8-CH3 10-CH3 12-OCH3 14-CH3 17-OCH3 1′-OCH3 1-NH− 11-OH 4′-OH
63.6 22.3 13.9 56.4 156.2 12.4 18.5 56.5 18.4 61.4 59.8
4.32, dqd (9.6, 6.6, 3.0) 1.26, d (6.6) 1.96, d (0.6) 3.22, s 1.34, 1.05, 3.33, 0.62, 4.07, 3.51, 7.91,
s d (6.6) s d (7.2) s s s
brd (9.0) m m m m m d (7.2)
5.20, d (10.2) 2.36, m 3.68, dd (9.0, 3.0) 3.06, overlap
31.4
1.80, m
28.8 29.6
129.3 156.6 181.5 119.6 143.3 182.7 81.0 207.9 48.3
2.85, dd (19.2, 9.6) 4′
5.90, 2.25, 2.17, 1.37, 1.25, 3.84, 5.01,
72.6 79.4
2.51, dd (13.2, 4.2) 16 17 18 19 20 21 1′ 2′ 3′
δH, mult. (J in Hz)
63.7 22.3 13.5 59.4 155.9 11.8 18.0 56.8 17.5 61.4 59.3
0.82, m 2.19, m 2.74, dd (12.6, 3.5) 2.52, dd (12.6, 3.5)
5.21, s 3.09, dd (18.6, 2.4) 2.77, dd (18.6, 9.6) 4.28, dqd (9.6, 6.6, 2.4) 1.28, d (6.6) 1.87, s 3.51, s 1.45, 1.12, 3.37, 0.62, 4.10, 3.51, 7.82,
s d (6.6) s d (6.6) s s s
Compound 2 showed very similar IR, MS, and NMR spectral features to those of 1, except that its NMR resonances assigned to two aliphatic methylenes (CH2-4, CH2-5) replaced the resonances for an sp2 hybrid disubstituted double-bond unit in 1 [δH 6.37 (dd, J = 12.0, 11.5 Hz, H-4), 5.32 (dd, J = 11.5, 10.2 Hz, H-5)]. The COSY correlations of H-3/H2-4/H2-5/H-6 and HMBC correlations from H-4 and H-5 to C-3 and C-6, in combination with the shifts of these proton and carbon B
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absent in GDM, originated mainly from its C-1′. As shikonin (1′R) showed a positive CE at 495 nm while 1 displayed a negative CE at 415 nm, the absolute configuration of C-1′ in 1 might be S. To determine the absolute configuration of C-1′ in 1 by ECD, a conformational analysis using the MMFF94 molecular force field was pursued, arbitrarily starting with the C-1′ Renantiomer of 1. For the different geometries of 1 and GDM, which were extracted from the trajectory of motion, the single ECD spectra were calculated by semiempirical ZINDO. Subsequent summing up of these ECD spectra provided the calculated overall ECD curves for the C-1′ R-enantiomer of 1 and GDM (Figure 3). As the ECD spectrum of the C-1′ Renantiomer of 1 and the experimental CD spectrum of 1 are opposite near 415 nm, the C-1′ of 1 may possess an Sconfiguration.
resonances, revealed that 2 was the 4,5-dihydrogenated analogue of 1. Thus, 2 was determined to be 4,5-dihydro-19(4′-hydroxy-1′-methoxy-2′-oxopentyl)geldanamycin, as expected. The NMR data of 2 are summarized in Table 1. Compared to GDM, the aliphatic substituent at C-19 of 1 (and 2) contains two chiral carbons at C-1′ and C-4′, respectively. Compound 2 was employed to elucidate the absolute configuration of C-4′ with sec-OH by the modified Mosher’s method,26,27 as more pure 2 was available. N,N′-Dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP) were applied to inhibit racemization of methoxyphenylacetic acid (MPA) in the esterification process.28 Ester derivatives of 2 with (R)- or (S)-MPA (2R, 2S) were prepared. While two (R)-MPA groups coupled to 2, one at C-4′ and the other at C-11 (the chiral carbon with sec-OH in the ansa-chain of the molecule), only one (S)-MPA group coupled to 2; the downfield shift of the C-4′ proton from δH 4.28 to δH 5.54 suggested that the (S)-MPA group combined with C-4′ of 2. From the MPA determination rule based on the Δδ values (Figure 2), the absolute configuration of C-4′ in 2 was established as R. The absolute configuration of C-4′ in 1 must also be R.
Figure 3. Calculated ECD spectra of 1 and GDM by semiempirical ZINDO.
The above result also proved that the negative CE at 415 nm came from the C-1′ linked to the benzoquinone moiety of 1, so the fragment molecule 1F was designated for the following precise ECD calculations using TDDFT methodology to further confirm the absolute configuration of C-1′ in 1. Six lowest energy conformers obtained after geometry optimization at the B3LYP/6-31G(d) level were considered for further analysis, which led to the UV and ECD spectra of six Boltzmann-averaged lowest energy conformers of 1F (UV, Supporting Information, Figure S31; ECD, Figure 4).29 The ECD spectrum showed similar CEs to the experimental CD spectrum of 1 [215 (−), 245 (+), 280 (−), 346 (+), and 529 (−) nm for the ECD; 220 (−), 248.5 (+), 271.5 (−), 303 (+), and 415 (−) nm for the experimental CD]. Thus, the absolute configuration of C-1′ in 1 was most probably S. The structure of 1 is 19-[(1′S,4′R)-4′-hydroxy-1′-methoxy2′-oxopentyl]geldanamycin, and the structure of 2 is 19[(1′S,4′R)-4′-hydroxy-1′-methoxy-2′-oxopentyl]-4,5-dihydrogeldanamycin (Chart 1). Compounds 1 and 2 showed thousands of times higher water solubility than GDM (15 400, 7705, and 2 μg/mL for 1, 2, and GDM, respectively). Compounds 1 and 2 displayed 50−80 times lower cytotoxicity against HepG2 cells (the IC50’s of 1, 2, and GDM were 14.6, 22.4, and 0.30 μM, respectively). More studies are necessary for a comprehensive evaluation of the potential of 1 and 2 as anticancer drug-lead candidates.
Figure 2. The absolute configuration of C-4′ in 2 is R by modified Mosher’s method.
The absolute configuration of C-1′ in 1 was studied by the experimental circular dichroism (CD) of 1, GDM, and shikonin (as a reference compound with a 1′ R substituent linked to the benzoquinone moiety, Chart 2) and then by comparing the experimental CD of 1 with the electronic CD (ECD) of 1F (a fragment molecule of 1, Chart 2). The CD spectra of 1, GDM, and shikonin were measured in methanol (Supporting Information, Figures S28, S29, and S30). The differential CD spectrum of 1 and GDM was almost the same as the CD spectrum of 1, indicating that all the Cotton effects (CEs) of 1, in particular the negative CE at 415 nm Chart 2. Chemical Structures of Shikonin and 1F
C
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preparation of 1. The same procedure was applied to the fermentation supernatant of the gdmP gene disruption mutant of S. hygroscopicus 17997 to give 2. 1: yellow, amorphous powder; CD (c 2.9 × 10−4 M, CH3OH) λmax (Δε) 415 (−3.4) nm, 303 (+15.3) nm, 271.5 (−6.3) nm, 248.5 (+8.7) nm, 220 (−6.2) nm; IR (KBr) νmax 3458, 2966, 2934, 2829, 1716, 1681, 1651, 1596 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 713.33 [M + Na]+; HRMS m/z 713.32567 [M + Na]+ (calcd for C35H50N2O12Na, 713.32615). 2: yellow, amorphous powder; IR (KBr) νmax 3469, 3351, 2964, 2933, 2832, 1720, 1655, 1608 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 715.39 [M + Na]+; HRMS m/z 715.34326 [M + Na]+ (calcd for C35H52N2O12Na, 715.34180). MPA Esters 2R and 2S. To a solution of 2 (6.92 mg, 0.01 mmol) and MPA (6.65 mg, 0.04 mmol) in CH2Cl2 (1.0 mL), DCC (8.25 mg, 0.04 mmol) and DMAP (4.9 mg, 0.04 mmol) were added, and the resultant mixture was stirred at room temperature under nitrogen protection for 24 h. It was then separated by normal-phase silica solid phase extraction and further purified by semipreparative HPLC [Agilent Zorbax SB C18 250 mm × 10 mm, CH3OH−H2O (55:45− 100:0)], obtaining 2R or 2S. 2R: yellow, amorphous powder; ESIMS m/z 1005.86 [M + NH4]+. 2S: yellow, amorphous powder; ESIMS m/z 857.90 [M + NH4]+. Cytotoxicity Assay. Compounds 1 and 2 were evaluated for cytotoxicity against HepG2 cells by the MTT assay. The assays were conducted with different concentrations of the compounds ranging from 0.5 to 300 μM (dissolved in DMSO with a final concentration of 0.1%). Computational Section. Conformational searches were carried out via the MOE ver. 2009.10 (Chemical Computing Group, Canada) software package using the MMFF94 molecular mechanics force field calculation followed by optimization at the B3LYP/6-31G(d) level. Six lowest electronic transitions were calculated using both dipole- length and dipole-velocity computed rotational strengths. ECD spectra were stimulated using a Gaussian function with a half-bandwidth of 0.5 eV. Equilibrium populations of conformers at 298.15 K were calculated from their relative free energies (ΔG) using Boltzmann statistics. The overall ECD spectra were then generated according to Boltzmann weighting of each conformer. All quantum computations were performed using the Gaussian03 package.30
Figure 4. Calculated ECD spectra of 1F using TDDFT, experimental CD spectrum of 1, and experimental CD differential spectrum of 1 and GDM.
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EXPERIMENTAL SECTION
General Experimental Procedures. IR spectra were acquired on a Nicolet 5700 FTIR microscope spectrometer. The 1H NMR (600 MHz) and 13C NMR (150 MHz) of 1 and 2 were recorded in CDCl3 in a Norell 3 mm nuclear tube with a Varian VNS-600 spectrometer. In elucidation of the sec-OH of C-4′ in 2, the 1H NMR (600 MHz) of 2, 2R, and 2S were analyzed in CDCl3 in a Norell 3 mm nuclear tube on a Varian VNS-600 spectrometer. Chemical shifts given in ppm were referenced to the solvent signals: δH/C 7.26/77.0 for CDCl3. ESIMS and HRMS were analyzed by LTQ Orbitrap XL from Thermo Fisher Scientific. Flash chromatography was performed on an EZ Purifier from Lisure Science (Suzhou) Co., Ltd., with a UV detector. Analytical and semipreparative HPLC was conducted on a Shimadzu LC-20AT or HPLC system with a photodiode array detector. The primary preparative HPLC separation was accomplished on a Shimadzu Prominence preparative HPLC system with LC-20AP pump and SPD20AV UV−vis detector. The ECD spectra of shikonin, GDM, and 1 were recorded on a JASCO J-815 spectropolarimeter. The spectra were average-computed over three instrumental scans and corrected by baseline subtraction obtained from a measurement of the same solvent used (CH3OH). Fermentation. S. hygroscopicus 17997, a wild-type GDM producer, was isolated from Chinese soil at the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. Frozen stock spores of S. hygroscopicus 17997 were thawed and spread onto ISPII medium plates (8.5 cm diameter; medium composition: 0.4% yeast extract, 1.0% malt extract, 0.4% glucose, and 1.5% agar powder) and incubated at 28 °C for 8−10 days for sporulation. Then slices of the seed culture were picked up and inoculated into the fermentation medium (2% starch, 0.5% cotton seed meal, 0.5% glucose, 1.0% cornsteep liquor, 0.5% dried yeast, 0.2% CaCO3) in 500 mL flasks at 28 °C for 96 h on a rotary shaker (200 rpm). The gdmP gene disruption mutant of S. hygroscopicus 17997 was cultured in the same way as its wild-type strain. Extraction and Isolation. The fermentation supernatant of S. hygroscopicus 17997 was extracted with an equal volume of EtOAc two or three times. The extract was concentrated under reduced pressure at room temperature, then fractionated by silica gel chromatography (2.5 cm × 80 cm; petroleum ether−EtOAc from 60:40 to 0:100 (v/v), stepwise elution). The fraction eluted by petroleum ether−EtOAc (20:80, v/v) was then subjected to Sephadex LH-20 chromatography (1.5 cm × 100 cm) to afford the crude preparation of 1. The crude preparation was refined by preparative HPLC [Shim-pack RPC-ODS, 20 mm × 250 mm; MeOH−water, 60:40 (v/v), 5.0 mL/min] and then semipreparative HPLC (Shimadzu LC-20ATvp, Shimadzu International Trading Co., Ltd., Beijing, China; Agilent ZorBax SBC18, 5 μm, 9.4 mm × 250 mm, Agilent Technologies Co. Ltd., Beijing, China; MeOH−water, 60:40 (v/v), 2.5 mL/min), yielding the pure
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ASSOCIATED CONTENT
S Supporting Information *
MS, IR, and NMR spectra of 1 and 2; experimental CD spectra of GDM, 1, and shikonin. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86-10-63165283. Fax: +86-10-63017302. E-mail:
[email protected] or
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (81172964), National Mega-project for Innovative Drugs (2012ZX09301002-001-016, 2012ZX09301002-003), and the Fundamental Research Funds for the Central Universities (2012N09).
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REFERENCES
(1) Sasaki, K.; Rinehart, K. L., Jr.; Slomp, G.; Grostic, M. F.; Olson, E. C. J. Am. Chem. Soc. 1970, 92, 7591−7593. (2) Johnson, R. D.; Haber, A.; Rinehart, K. L., Jr. J. Am. Chem. Soc. 1974, 96, 3316−3317. (3) BeBoer, C.; Dietz, A. J. Antibiot. 1976, 29, 1182−1188.
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