Pyrimidine Nucleosides from Streptomyces sp. SSA28 | Journal of

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Pyrimidine Nucleosides from Streptomyces sp. SSA28 Cheng-Dong Xu, Hao-Jian Zhang, and Zhong-Jun Ma* Institute of Marine Biology and Pharmacology, Ocean College, Zhejiang University, Zhoushan 316021, People’s Republic of China

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S Supporting Information *

ABSTRACT: Eleven new pyrimidine nucleosides (1−11) and 12 known analogues (12−23) were isolated from the marine-derived Streptomyces sp. SSA28. All of the new structures were elucidated by extensive NMR spectroscopic analysis and HRESIMS data. The absolute configurations of compound 1 were determined by X-ray diffraction. The configurations of 2−16 were investigated by ECD calculations. Compounds 11−16 showed cytotoxicity against HCT-116 human colon cancer cell lines with IC50 values from 0.39 ± 0.03 to 6.63 ± 0.47 μM.

micetin, a disaccharide nucleoside antibiotic, was first reported in 1953 from a soil-derived Streptomyces.1,2 During the past decades, 10 amicetin-like nucleosides had been reported from nature, including bamicetin,3 plicacetin,3 oxamicetin,4 norplicacetin,5 oxyplicacetin,6 cytosaminomycin B,7 streptcytosine A,8 SF2457,9 40551-D, and 40551-H.10 Previous studies showed amicetin-type nucleosides exhibit activity against mycobacteria and Gram-positive bacteria.9,10 The anticoccidial activity of cytosaminomycin A and antituberculosis activity of 40551-F indicated that amicetin analogues without PABA were bioactive.7,10 Furthermore, the reported unnamed amicetin-like primidine nucleoside (21) which was cytotoxic against ACHN, Panc-1, and HCT 116 cells brought the potential anticancer bioactivity to our notice.11 In this study, a marine-derived Streptomyces sp. SSA28 was investigated for cytotoxic products with novel structures, and 11 new amicetin-like pyrimidine nucleosides (1−11) and 12 known compounds including streptcytosine B−E (17−20),8 amicetin (23),2 two unnamed pyrimidine nucleosides (21 and 22),11,12 and five configuration undetermined disaccharyl analogues, 40551-D (15), 40551-F (14), 40551-G (16), 40551-K (13), and 40551-L (12) (Figure 1), were discovered.10 Compounds 11−16 had significant cytotoxicity against HCT-116 cancer cell lines with IC50 value less than 1 μM. Herein, the isolation, structural elucidation, and bioactivity evaluation of these secondary metabolism products are reported.

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that compound 1 had a hydroxy-substituted isopropyl compared with streptcytosine E, which was further supported by HMBC and COSY correlations (Figure 2). In the NOESY spectrum (Figure 3), the correlation of H-1′ and H-5′ indicated they had the same orientation, while the large coupling constant (J = 9.0) between H-4′ and H-5′ suggested a different orientation. Thus, the relative configuration of compound 1 was determined. The absolute configuration of compound 1 was investigated by X-ray diffraction. Colorless crystals of 1 were grown in a mixture of isopropanol, n-butanol, and water (7:7:1). X-ray diffraction analysis performed with Cu Kα radiation revealed the absolute configuration of the sugar unit as 1′R, 4′S, and 5′S (Figure 4), which was further confirmed by electronic circular dichroism (ECD) calculations (Figure 5). Streptcytosine G (2) had a molecular formula of C15H21O5N3, as deduced from its HRESIMS data, indicating seven indices of hydrogen deficiency. The 1H and 13C NMR data (Tables 1 and 3) of compound 2 were almost identical with the known compound strepcytosine D (19),8 except that C-3′ was replaced by a hydroxy in 2. The relative configuration of 2 was determined by NOESY correlations (H-1′/H-3′/H5′) and the large coupling constant of H-4′/H-5′ (J = 9.0 Hz) (Figure 3). Fortunately, the hydrolysate of compound 2 (Figure 4) formed suitable crystals by the same solvent of compound 1. Thus, the absolute configuration of the hydrolysate product, the amicetosylcytosine unit, was determined as shown by X-ray diffraction analysis, and the new compound 2 was named streptcytosine G (Figure 4). Streptcytosine H−K (3−6) had molecular formulas of C12H17O4N3, C13H19O4N3, C14H21O4N3, and C16H25O4N3 judged by HRESIMS data, respectively. Their 1D NMR data

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RESULTS AND DISCUSSION Streptcytosine F (1) was obtained as colorless needle crystals. HRESIMS showed it had a molecular formula of C15H23O5N3, determining an index of hydrogen deficiency of six. Detailed analysis of the 1D NMR data (Tables 1 and 3) indicated that 1 was an analogue of streptcytosine E.8 The main difference was © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 1, 2019

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DOI: 10.1021/acs.jnatprod.9b00260 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Structures of compounds 1−23: new compounds (1−11); known compounds (12−23).

Table 1. 1H NMR (600 MHz) Data of Compounds 1−6 (δH, J in Hz) no.

1a

2a

3a

4a

5a

6a

5 6 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′

7.48, d (7.6) 8.12, d (7.6) 2.58, s

7.57, d (7.6) 8.10, d (7.6) 5.94, m

7.45, d (7.5) 8.10, d (7.5) 2.17, s

7.48, d (7.5) 8.10, d (7.5) 2.46, q (7.5) 1.15, t (7.5)

1.31, s 1.31, s

1.96, d (1.3) 2.22, d (1.3)

7.49, d (7.6) 8.10, d (7.6) 2.68, m 1.17, d (2.1) 1.18, d (2.1)

5.70, dd (10.1, 2.2) 2.11, m 1.66, overlap 2.14, m 1.66, overlap 3.27, m 3.57, dq (9.0, 6.2) 1.32, d (6.2)

5.78, dd (10.9, 2.2) 2.33, m 1.62, d (10.9) 3.69, ddd (11.2, 9.2, 4.9) 3.06, t (9.2) 3.50, dq (9.2, 6.2) 1.36, d (6.2)




7.47, d (7.5) 8.09, d (7.5) 2.45, t (7.5) 1.55, m 1.60, overlap 0.93, d (6.4) 0.93, d (6.4) 5.69, dd (7.8, 2.3) 2.10, m 1.66, overlap 2.15, m 1.66, overlap 3.27, m 3.50, dq (9.2, 6.2) 1.33, d (6.2)

a,b

Recorded in methanol-d4 and DMSO-d6, respectively.

(Tables 1 and 3) were remarkably similar to those of compound 1. The main differences between them were that the side chains connected at C-4 in 1 were replaced by acetamide, propanamide, isobutyramide, or isovaleramide in 3−6, respectively, which was also supported by key COSY and HMBC correlations (Figure 2). The NOESY data of compounds 3−6 (Figure 3) showed that the sugar moiety shared the same relative configuration with 1. In addition, their experimental ECD curves (see Supporting Information (SI) Figures S30, S40, S50, S60) were identical to those of compound 1, indicating the same absolute configurations. Finally, the structures of compounds 3−6 were determined as

shown (Figure 1) and named streptcytosines H (3), I (4), J (5), and K (6). Streptcytosines L (7) and M (8) had the same molecular formula of C16H23O4N3 as indicated by HRESIMS analysis, showing that both had one less degree of unsaturation than streptcytosine K (6). Analysis of their 1D and 2D NMR data suggested that 7 and 8 were analogues of compound 6. The differences were the presence of a Δ9 or Δ10 double bond in 7 and 8, respectively, corroborated by the HMBC cross-peaks. The absolute configurations were also determined as 1′R, 4′S, and 5′S by NOESY experimentation (Figure 3) and by comparing their experimental ECD spectra (see SI Figures S70, S80) with compound 1. B



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Table 2. 1H NMR (600 MHz) Data of Compounds 7−11 (δH, J in Hz) 7b

no. 5 6 9 10 11 12 13 14 15 17 18 19 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″

7.21, 8.07, 3.14, 5.26,

d (7.5) d (7.5) d (7.2) t (7.2)

1.60, s 1.69, s

5.61, 1.88, 2.00, 3.11, 3.38, 1.19,

dd (10.7, 2.3) m; 1.65, m m; 1.52, m m dq (9.0, 6.1) d (6.1)

8a 8.11, 7.55, 6.12, 7.04, 2.52, 1.10, 1.10,

5.70, 2.10, 2.15, 3.27, 3.50, 1.33,

d (7.5) d (7.5) dd (15.5, 1.5) dd (15.5, 6.7) m d (6.8) d (6.8)

dd (10.1, 2.4) m; 1.66, overlap m; 1.66, overlap m dq (9.1, 6.1) d (6.1)

9a

10a

7.45, d (7.5) 8.09, d (7.5) 3.75, s

7.60, d (7.5) 8.12, d (7.5)

7.32, 7.32, 7.25, 7.32, 7.32,

5.70, 2.10, 2.14, 3.27, 3.47, 1.32,

overlap overlap m overlap overlap

dd (10.0, 2.1) m; 1.65, overlaap m; 1.65, overlap m dq (9.3, 6.1) d (6.1)

7.80, d (9.0) 6.63, d (9.0) 6.63, d (9.0) 7.80, d (9.0) 4.16, 2.17, 1.41, 5.72, 2.10, 2.15, 3.28, 3.50, 1.33,

q (7.1) s d (7.1) dd (10.2, 2.1) m; 1.69, overlap m; 1.69, overlap m dq (9.2, 6.0) d (6.0)

11a 7.46, 8.10, 2.46, 1.56, 1.60, 0.93, 0.93,

d (7.6) d (7.6) t (7.7) overlap m d (6.5) d (6.5)

5.75, 2.15, 2.37, 3.42, 3.74, 1.37, 5.01, 3.54, 3.95, 3.12, 4.09, 1.46, 3.01, 3.01,

dd (9.9, 2.6) m; 1.68, overlap m; 1.68, overlap m dd (9.0, 6.1) d (6.1) d (4.0) dd (9.2, 4.0) dd (10.1, 9.2) t (10.1) dq (10.1, 6.2) d (6.2) s s

a,b


of the disaccharide unit as 1′R, 4′S, 5′R, 1″R, 2″R, 3″S, 4″R, 5″R. According to the literature,10 compounds 12−16 (40551-L (12), 40551-K (13), 40551-F (14), 40551-D (15), and 40551G (16)) are known products whose configurations were undetermined.10 Herein, the configurations of these five compounds were determined by NOESY spectra and ECD calculations. Similar to compound 11, their NOESY spectrum (Figure 3) also showed correlations of H-1′/H-5′, H-4′/H-1″, H-2″/H-4″, and H-3″/H-5″, which indicated they shared the same relative configuration of amosamine and amicetose. The identical experimental ECD curves (Figure 5) suggested the same absolute configurations of the sugar as 1′R, 4′S, 5′R, 1″R, 2″R, 3″S, 4″R, 5″R, compared to compound 11 (Figure 4). The biosynthesis pathway of amicetin (23) by Zhang13,14 revealed 21 genes are involved in the synthesis of amicetose, amosamine, cytosine, PABA-CoA, and their interrelated linkage. It revealed that the glycosylation of amicetose and amosamine and the amide bond formation of cytosine and PABA were two key steps, but the sequence of these two steps remained controversial. Inspired by the similar structure to amicetin, compounds isolated in this study are proposed to share similar biosynthesis pathways (SI Figure S161). Furthermore, comparison of compounds 6, 8, 17, 18, and 20 with compounds 12, 11, 21, 14, and 13 suggests that the glycosylation between amicetose and amosamine might occur before the amidation at N-7 of cytosine. Compounds 11, 12, and 16 showed better cytotoxicity against the HCT-116 human colon cancer cell line than known compound 21,11 with IC50 values of 0.39 ± 0.03, 0.78 ± 0.05, and 0.89 ± 0.06 μM, respectively. Compounds 13, 14, and 15 exhibited moderate activity, with IC50 values of 2.14 ± 0.20,

The molecular formula C18H21O4N3 of streptcytosine N (9) was given by HRESIMS. The characteristic proton chemical shifts at δH 7.25−7.32 show the appearance of a monosubstituted benzene ring. According to key HMBC cross-peaks (Figure 2) of H-9 with C-8, C-10, C-11, and C-15, the benzene was attached to C-9. The NOESY data (Figure 3) and ECD curve (see SI Figure S90) supported its absolute configuration as 1′R, 4′S, and 5′R. Streptcytosine O (10) had a molecular formula of C20H26O5N4 based on HRESIMS data. Two 13C NMR signals (δC 131.4, 112.8) and two characteristic 1H NMR signals [δH 7.8 (2H, d, J = 8.9 Hz); δH 6.63 (2H, d, J = 9.0 Hz)] (Tables 2 and 3) indicated that there was a para-substituted benzene ring in the side chain at N-7. Further HMBC correlations (Figure 2) of H-16 to C-17/C-19 and H-18 to C-17 suggested a carbonyl C-17 connected with a methyl (C-18) and a methine (C-16) which was linked to another methyl (C-19). NOESY data (Figure 3) and ECD calculation (see SI Figure S100) showed the absolute configuration was the same as that of compound 9. The molecular formula of cytosaminomycin E (11) was determined to be C24H40N4O7 by HRESIMS. Comparing the 1D NMR data (Tables 2 and 3) with those of compound 6, cytosaminomycin E (11) had the same structure except for an amosamine (4,6-dideoxy-4-dimethylamine-D-glucose) connected with an amicetose by an α-(1→4)-glycoside bond. The NOESY data, which showed correlations of H-1′/H-5′, H4′/H-1″, H-2″/H-4″, and H-3″/H-5″ and coupling constants of H-4′/H5′ and H-4″/H-5″, determined the relative configuration of compound 11 (Figure 3). Experimental ECD curves of compound 11 and amicetin gave similar fitted curves (Figure 5), confirming the same absolute configuration C


Journal of Natural Products Table 3.

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C NMR (150 MHz) Data of Compounds 1−11 (δC, Type)

no.

1a

2a

3a

4a

5a

6a

7b

8a

9a

10a

11a

2 4 5 6 8 9 10 11 12 13 14 15 16 17 18 19 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″

157.3 163.8 98.3 146.4 173.6 51.1 70.4 29.6 29.6

157.4 164.9 98.5 145.8 167.7 118.8 159.6 27.8 20.5

157.5 164.3 98.3 146.2 173.0 24.5

157.5 164.3 98.3 146.1 176.5 31.3 9.2

157.5 164.5 98.4 146.2 179.8 37.2 19.4 19.4

157.5 164.3 98.4 146.2 176.1 36.2 34.9 28.9 22.7 22.7

154.0 162.4 95.7 145.6 172.3 36.2 116.8 134.7 18.0 25.5

157.4 164.6 98.6 146.1 167.6 121.7 156.8 32.3 21.6 21.6

157.4 164.3 98.3 146.3 173.7 44.7 135.9 130.4 129.7 128.2 129.7 130.4

157.5 165.0 98.7 146.0 168.4 153.4 131.4 112.8 121.6 112.8 131.4

157.6 164.1 98.4 146.1 176.1 36.2 34.8 28.9 22.6 22.6

84.7 31.6 32.3 71.6 80.4 18.5

82.6 39.8 72.1 77.9 76.9 18.3

84.7 31.6 32.3 71.6 80.4 18.5

84.7 31.6 32.3 71.3 80.4 18.5

84.7 31.6 32.3 71.6 80.4 18.5

84.7 31.6 32.3 71.6 80.4 18.5

82.2 29.9 31.4 69.7 78.6 18.3

84.7 31.6 32.3 71.6 80.4 18.5

84.7 31.6 32.3 71.6 80.4 18.5

59.3 212.0 25.5 18.5 84.7 31.6 32.3 71.6 80.4 17.3

84.6 30.9 28.0 76.6 78.2 19.2 96.7 73.9 67.9 72.0 64.0 19.0 42.0 42.0

a,b


6.45 ± 0.57, and 6.63 ± 0.47 μM (Table 4), while the monosaccharide nucleosides (1−10) showed almost no cytotoxicity. This results suggest that amosamine plays an important role in cytotoxicity, and the different cytotoxicities of compounds 11−16 indicated that the aliphatic chain at N-7 also might influence cytotoxicity.

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water) and stored in 20% aqueous glycerite. Identification work was conducted by Takara Biotechnology (Dalian, China), comparing the 16S rDNA sequence with the GenBank database after PCR amplification. The sequence was submitted to NCBI GenBank (accession no. MK611756). Cultivation of SSA28. Spores of the strain were inoculated into 500 mL Erlenmeyer flasks containing 250 mL of liquid-Gause medium to produce a seed inoculum, incubating for 7 days at 28 °C on a shaker (180 rpm). A 10 mL amount of this culture was added into each of 500 mL Erlenmeyer flasks that contained 40 g of rice (boiled with 60 mL of seawater), and these flasks were kept at 28 °C for one and a half months. In total, 12 kg of rice culture was prepared. Extraction and Isolation. The fermentation material was extracted with 500 mL of ethyl acetate added to each flask. Twelve grams of extract was produced after drying the organic phase by rotary evaporator. The oil phase was removed by liquid−liquid separation between petroleum ether and 80% aqueous MeOH. The MeOH fraction (6.3 g) was subjected to silica gel chromatography (120 g, 300−400 mesh) eluting with a mixture of CH2Cl2−MeOH (70:1, 50:1, 30:1, 15:1, 10:1, 5:1, 2:1, and 0:1, 1.5 L each) to yield 17 fractions (A−Q). Fraction G (120 mg) was purified by semipreparative HPLC (MeCN−H2O, 18%, flow rate 10 mL/min) to give compound 1 (10 mg, tR = 10 min). Fraction K was purified by preparation HPLC (MeCN−H2O, 16%) to provide 2 (2.1 mg, tR = 49 min). By the same method (MeCN+0.05%TFA−H2O, 15%, flow rate 10 mL/min), compounds 3 (2.4 mg, tR = 6 min), 4 (4 mg, tR = 10 min), 5 (3 mg, 29 min), 6 (7.3 mg, tR = 91 min), 7 (5 mg, tR = 76 min), 8 (2.3 mg, tR = 83 min), 9 (8 mg, tR = 78 min), and 10 (5 mg, tR = 62 min) were obtained from fraction D. Fraction M was fractionated by MPLC (MeCN−H2O, 20−55%, 60 min, flow rate 10 mL/min) to give 11 subfractions (M1−M11). Purified by semi-

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined with a Rudolph API-35201 automatic polarimeter. 1D and 2D NMR were recorded on a JEOL JNM-ECZR instrument or Bruker AV III instrument using tetramethylsilane as the standard substance. The chemical shifts (δ) were acquired in ppm with reference to the solvent signals. HRESIMS data were recorded with an Agilent 1260 HPLC-6230 TOF tandem spectrometer. HPLC analysis was performed with a Shimadzu DGU 20A5 system equipped with an Agilent Pursuit XRs-C18 column (10 μm, 4.6 × 250 mm). Preparative HPLC was performed with a Shimadzu LC-20AP system equipped with an Agilent Pursuit C18 column (10 μm, 21.2 × 250 mm). UV and IR spectra were measured by a Shimadzu UV-1800 spectrophotometer and a Thermo Fisher Nicolet IS10 spectrophotometer. ECD spectra were collected on a JASCO J-1500-150ST. Column chromatography was performed on silica gel CC (300−400 mesh, Qingdao Haiyang Chemical Company) and on Sephadex LH-20 stationary phases. Strain Isolation and Identification. Strain SSA28 was isolated from a marine sediment sample from Shengsi Archipelago (Zhejiang Province, China). The strain was cultured on solid-Gause medium (20 g starch, 1 g KNO3, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g NaCl, 0.01 g FeSO4·7H2O, 20 g agar, 25 g artificial sea salt per liter D



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Figure 2. Key COSY (bold lines) and HMBC (arrows) correlations of compounds 1−16.

Figure 3. Key NOESY spectrum (imaginary lines) and coupling constants (full lines) of the sugar unit: (a) compounds 1 and 3−10; (b) compound 2; (c) compounds 11−16. cm−1; UV (MeOH) λmax (log ε) 207(4.61), 265 (4.68), 300 (4.43) nm; ECD (MeOH), λmax (Δε) 210 (+2.30), 243 (−7.55),307 (7.17) nm; 1H and 13C NMR data in Tables 1 and 2; HRESIMS m/z 324.1555 [M + H]+ (calcd for C15H21N3O5, 324.1554). Streptcytosine H (3): colorless oil; [α]20D +64 (c 0.05, MeOH); IR νmax 3234, 2932, 1714, 1648, 1565, 1493, 1392, 1309, 1245, 1089, 995, 786 cm−1; UV (MeOH) λmax (log ε) 212 (4.68), 247 (4.50), 296 (4.17) nm; ECD (MeOH), λmax (Δε) 207 (+7.51), 223 (−14.03), 265 (+7.34), 300 (+6.30) nm; 1H and 13C NMR data in Tables 1 and 2; HRESIMS m/z 268.1295 [M + H]+ (calcd for C12H17N3O4, 268.1292). Streptcytosine I (4): white solid; [α]20D +52 (c 0.05, MeOH); IR νmax 3234, 2975, 1648, 1561, 1492, 1392, 1337, 1311, 1269, 1155, 1089, 1057, 787 cm−1; UV (MeOH) λmax (log ε) 212 (4.74), 248

preparative HPLC (MeCN−H2O, 0.05% TFA), subfraction M6 gave compound 13 (2.8 mg), subfraction M8 gave compound 14 (2.8 mg), subfraction M9 gave compound 12 (3.1 mg), subfraction M10 gave compound 11 (1.8 mg), and subfraction M11 gave compounds 16 (2.1 mg) and 15 (2.0 mg), separately. Streptcytosine F (1): white solid; [α]20D +332 (c 0.05, MeOH); IR νmax 3339, 1648, 1562, 1490, 1436, 1381, 1337, 1308, 1268,1225, 1186, 1089, 1057, 787 cm−1; UV (MeOH) λmax (log ε) 212 (4.77), 249 (4.73), 300 (4.39) nm; ECD (MeOH), λmax (Δε) 206 (+13.37), 224 (−23.55), 265 (+12.36), 301(+10.96) nm; 1H and 13C NMR data in Tables 1 and 2; HRESIMS m/z 326.1714 [M + H]+ (calcd for C15H23N3O5, 326.1710). Streptcytosine G (2): colorless oil; [α]20D +56 (c 0.05, MeOH); IR νmax 3292, 1639, 1563, 1489, 1432, 1395, 1330, 1229, 1131, 1074, 787 E



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Figure 4. (a) ORTEP drawing of compound 1; (b) ORTEP drawing of compound 2. (4.67), 298 (4.37) nm; ECD (MeOH), λmax (Δε) 207 (+10.28), 222 (−19.56), 262 (+9.65), 300 (+9.24) nm; 1H and 13C NMR data in Tables 1 and 2; HRESIMS m/z 282.1452 [M + H]+ (calcd for C13H19N3O4, 282.1448). Streptcytosine J (5): white solid; [α]20D +100 (c 0.05, MeOH); IR νmax 3335, 2973, 2933, 1647, 1565, 1489, 1387, 1309, 1270, 1184, 1089, 1056, 994, 787 cm−1; UV (MeOH) λmax (log ε) 211 (4.65), 248 (4.58), 298 (4.21) nm; ECD (MeOH), λmax (Δε) 208 (+6.76), 224 (−20.35), 268 (+9.85), 300 (+8.37) nm; 1H and 13C NMR data in Tables 1 and 2; HRESIMS m/z 296.1602 [M + H]+ (calcd for C14H21N3O4, 296.1605). Streptcytosine K (6): white solid; [α]20D +112 (c 0.05, MeOH); IR νmax 3338, 2955, 1648, 1563, 1490, 1380, 1337, 1309, 1269, 1088, 994, 787 cm−1; UV (MeOH) λmax (log ε) 210 (4.74), 248 (4.67), 299 (4.27) nm; ECD (MeOH), λmax (Δε) 207 (+8.05), 227 (−19.57), 268 (+11.23) nm; 1H and 13C NMR data in Tables 1 and 2; HRESIMS m/z 346.1742 [M + H]+ (calcd for C16H25N3O4, 346.1737). Streptcytosine L (7): white solid; [α]20D +96 (c 0.05, MeOH); IR νmax 3224, 2967, 2894, 1706, 1678, 1620, 1568, 1487, 1440, 1348, 1276, 1187, 1153, 1079, 992, 803 cm−1; UV (MeOH) λmax (log ε) 211 (4.64), 248 (4.57), 299 (4.26) nm; ECD (MeOH), λmax (Δε) 209 (+8.83), 225 (−17.40), 267 (+8.08), 302 (+8.45) nm; 1H and 13 C NMR data in Tables 1 and 2; HRESIMS m/z 322.1757 [M + H]+ (calcd for C16H23N3O4, 322.1761). Streptcytosine M (8): white solid; [α]20D +104 (c 0.05, MeOH); IR νmax 3253, 2958, 1678, 1493, 1316, 1203, 1135, 1089, 1053, 800, 722 cm−1; UV (MeOH) λmax (log ε) 217 (4.72), 262 (4.80), 303 (4.44) nm; ECD (MeOH), λmax (Δε) 210 (+1.60), 239 (−9.05), 278 (+6.88), 309 (+6.12) nm; 1H and 13C NMR data in Tables 1 and 2; HRESIMS m/z 322.1760 [M + H]+ (calcd for C16H23N3O4, 322.1761). Streptcytosine N (9): white solid; [α]20D +48 (c 0.05, MeOH); IR νmax 3342, 1648, 1563, 1490, 1438, 1337, 1307, 1269, 1186, 1088,1056, 994, 786 cm−1; UV (MeOH) λmax (log ε) 203 (4.51), 248 (4.32), 300 (3.91) nm; ECD (MeOH), λmax (Δε) 209 (+5.75), 223 (−22.59), 265 (+11.86), 304 (+8.73) nm; 1H and 13C NMR data in Tables 1 and 2; HRESIMS m/z 344.1599 [M + H]+ (calcd for C18H21N3O4, 344.1605). Streptcytosine O (10): white solid; [α]20D +80 (c 0.05, MeOH); IR νmax 3345, 1651, 1603, 1560, 1482, 1336, 1255, 1185, 1085, 786 cm−1; UV (MeOH) λmax (log ε) 205 (4.81), 254 (4.57), 330 (4.85) nm; ECD (MeOH), λmax (Δε) 203 (+4.87), 230 (−10.58), 272

(+4.58), 306 (+5.04) nm; 1H and 13C NMR data in Tables 1 and 2; HRESIMS m/z 415.1977 [M + H]+ (calcd for C21H26N4O5, 415.1976). Cytosaminomycin E (11): yellowish oil; [α]20D +100 (c 0.05, MeOH); IR νmax 3253, 2958, 1678, 1573, 1493, 1316, 1203, 1135, 1053, 800, 722 cm−1; UV (MeOH) λmax (log ε) 202 (4.41), 248 (4.23), 299 (3.91) nm; ECD (MeOH), λmax (Δε) 205 (+3.24), 224 (−6.6), 263 (+3.66), 298 (+2.51) nm; 1H and 13C NMR data in Tables 1 and 2; HRESIMS m/z 497.2967 [M + H]+ (calcd for C24H40N4O7, 497.2970). X-ray Crystal Data for Compound 1. Colorless crystal of C15H23N3O5, M = 325.36, orthorhombic, space group P212121 (no. 19), a = 9.06338(13) Å, b = 10.86547(13) Å, c = 17.5252(2) Å, V = 1725.84(4) Å3, Z = 4, T = 293.68(10) K, μ(Cu Kα) = 0.789 mm−1, Dcalc = 1.252 g/cm3, 13 705 reflections measured (9.578° ≤ 2θ ≤ 147.072°), 3437 unique (Rint = 0.0291, Rsigma = 0.0221), which were used in all calculations. The final R1 was 0.0413 (I > 2σ(I)) and wR2 was 0.1057 (all data). Crystallographic data for 1 (CCDC 1902243) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk. X-ray Crystal Data for the Hydrolysate of Compound 2. Colorless crystal for C10H15N3O3, M = 225.25, monoclinic, space group P21 (no. 4), a = 9.6749(2) Å, b = 10.4511(2) Å, c = 10.9170(2) Å, β = 98.643(2)°, V = 1091.32(4) Å3, Z = 4, T = 293.68(10) K, μ(Cu Kα) = 0.858 mm−1, Dcalc = 1.371 g/cm3, 16 623 reflections measured (8.192° ≤ 2θ ≤ 149.692°), 4328 unique (Rint = 0.0350, Rsigma = 0.0256), which were used in all calculations. The final R1 was 0.0278 (I > 2σ(I)) and wR2 was 0.0756 (all data). Crystallographic data for the hydrolysate of compound 2 (CCDC 1902242) can be obtained from the Cambridge Crystallographic Data Centre for free via www.ccdc.cam.ac.uk. ECD Calculations. ECD calculation data were compared with the experimental ECD result. The conformers with a Boltzmann population of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at the B3LYP/6-31+G(d,p) level in MeOH using the CPCM polarizable conductor calculation model. The theoretical calculation of ECD was conducted in MeOH using time-dependent density functional theory at the B3LYP/6311+G(d,p) level for all conformers of compound 1. Rotatory strengths for a total of 50 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University F



Article

Figure 5. (a) Experimental ECD and calculated ECD spectra of compounds 1, 11, and amicetin; (b) experimental ECD of compounds 11−16. Cytotoxicity Assay. Cytotoxicity toward the human colon cancer cell line HCT-116 was tested by the SRB method with adriamycin as positive control as described before.15 The details are given in the Supporting Information.

Table 4. Cytotoxicity Activities of 1−16 IC50 (μM) compound

HCT-116

1−10 11 12 14 15 13 16 adriamycin

>20 0.39 ± 0.03 0.78 ± 0.05 6.45 ± 0.57 6.63 ± 0.47 2.14 ± 0.20 0.89 ± 0.06 0.06 ± 0.01

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00260. 1D, 2D NMR, HRESIMS, IR, and UV spectra of new compounds and ECD spectra for compounds 1−16 (PDF) X-ray crystallographic data (CIF) X-ray crystallographic data (CIF)

of California San Diego, CA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV. G



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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Zhong-Jun Ma: 0000-0002-5825-5095 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Fundamental Research Funds for the Central Universities (No. 2018QNA4043).

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REFERENCES

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