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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Griseofamines A and B: Two Indole-Tetramic Acid Alkaloids with 6/5/6/5 and 6/5/7/5 Ring Systems from Penicillium griseof ulvum Yi Zang,† Grégory Genta-Jouve,‡ Yingyu Zheng,† Qing Zhang,† Chunmei Chen,† Qun Zhou,† Jianping Wang,*,† Hucheng Zhu,*,† and Yonghui Zhang*,† †

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ C-TAC, UMR 8638 CNRS, Faculté des Sciences Pharmaceutiques et Biologiques, Paris Descartes University, Sorbonne Paris Cité, 4 Avenue de l’Observatoire, 75006 Paris, France S Supporting Information *

ABSTRACT: Two novel indole-tetramic acid alkaloids griseofamine A (1) and griseofamine B (2)and (R)-N-(2methylbutanoyl)-L-tryptophan (3), were isolated from the fungus Penicillium griseofulvum. Compounds 1 and 2 feature a 6/5/6/5 and 6/5/7/5 tetracyclic ring systems formed by the fusion of an indole unit and a tetramic acid via a six or sevenmembered N-heterocyclic ring, respectively. The plausible biosynthetic pathways of 1−3 are proposed. Compound 1 shows a weak anti-inflammatory activity by inhibition of NO and TNF-α production.

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atural products containing a tetramic acid (pyrrolidine2,4-dione) nucleus are isolated from terrestrial and marine organisms.1 Among them, the 3-position of the tetramic acid ring can be substituted by an acyl moiety to afford 3-acyl tetramic acids, which are usually inseparable tautomeric mixtures detected in solution with Z-exo-enol and E-exo-enol form in a ratio.1−3 Those alkaloids exhibit a wide range of biological activities, including antibacterial and antitumoral activities.1 Indole-tetramic acid alkaloids represent a unique type that is structurally characterized by a indole and a 3-acyl tetramic acid scaffold.4−6 Over the past decades, only a few indole-tetramic acid alkaloid-related analogues have been explored and comprise an almost-invariable backbone.6 Herein, cultures of the fungus Penicillium griseofulvum grown on solid rice media was studied to yield two pure indoletetramic acid alkaloids possessing novel frameworks, called griseofamine A (1) and griseofamine B (2), together with (R)N-(2-methylbutanoyl)-L-tryptophan (3), which was first reported as a natural product. Griseofamine A (1) and griseofamine B (2) are the first alkaloids fused an indole unit to a tetramic acid scaffold via a six-membered or sevenmembered N-heterocyclic ring to generate a surprising 6/5/6/5 and 6/5/7/5 tetracyclic ring systems, respectively. Their absolute configurations are established by single-crystal X-ray diffraction (XRD) analysis, theoretical nuclear magnetic resonance (NMR), and electronic circular dichroism (ECD) calculation. All compounds were evaluated based on their antiinflammatory effect. The biogenetic origin will be discussed, hypothesizing a key origin from L-tryptophan for the skeleton. © XXXX American Chemical Society

Compound 1 was isolated as white powder with an [α]D20 of −108.0 (c 0.1, MeOH). The molecular formula of C22H24N2O3 with 12 degrees of unsaturation was deduced from the pseudomolecular ion peak at m/z 387.1688 [M + Na]+ in highresolution electrospray ionization mass spectrometry (HRESIMS). The infrared (IR) spectrum displayed the presence of hydroxyl group at 3331 cm−1 and carbonyl groups at 1714 and 1622 cm−1. The data analyses of 1H, 13C, and heteronuclear single quantum correlation (HSQC) NMR spectra (Table 1) indicated the presence of a substituted indole moiety with significant signals at δH 11.08 (1H, s, 1-NH), 7.16 (1H, d, 7.7 Hz, H-7), 6.96 (1H, dd, 7.7, 7.1 Hz, H-6), and 6.72 (1H, d, 7.1 Hz, H-5); δC 136.5 (C-8), 133.8 (C-2), 133.3 (C-4), 124.5 (C9), 121.4 (C-6), 118.9 (C-5), 109.3 (C-7), and 104.5 (C-3). An observed tetramic acid unit showed characteristic signals at δH 4.36 (1H, dd, 11.0, 5.4 Hz, H-11), 2.43 (3H, s, 19-CH3); δC 194.4 (C-12), 183.0 (C-18), 169.8 (C-14), 102.0 (C-13), 57.6 (C-11), 19.2 (C-19). The other signals displayed three methyl groups with two singlets at δH 1.71 (3H, 24-CH3) and 1.69 (3H, 23-CH3) and a doublet at δH 1.52 (3H, d, 6.7 Hz, 17CH3), two methylenes at δH 3.60 (2H, d, 6.7 Hz, H-20), 3.37 Received: February 17, 2018

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DOI: 10.1021/acs.orglett.8b00584 Org. Lett. XXXX, XXX, XXX−XXX

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addition, an isoprenyl fragment (−CH2−CHC(CH3)−CH3) was concluded by cross peaks between H-20 and H-21 in the 1 H−1H COSY and correlations from 23-CH3 and 24-CH3 to C21/C-22 in the HMBC spectrum. Further HMBC correlations from H-21 to C-4 and from H-20 to C-4/C-5/C-9 revealed the location of the isoprenyl group at C-4. Therefore, the planar structure of 1 was concluded as shown in Figure 1. The relative stereochemistry of 1 was deduced from coupling constants, the NOESY experiment, and X-ray crystallography. The sharing large coupling constant of 3JH‑11,H‑10β (11.0 Hz) indicated H-11 and H-10β was a trans-diaxial relationship while small coupling constant of 3JH‑11,H‑10α (5.4 Hz) was induced from H-11 and H-10α. In addition, observed key correlations between H-11/H-10α and H-11/17-CH3 in the nuclear Overhauser effect spectroscopy (NOESY) spectrum (Figure 1) showed that H-10α, H-11, and 17-CH3 were in a cofacial position on the ring C, while H-10β and H-16 were on the opposite side. The assignment of the relative stereochemistry of 1 was further confirmed by X-ray diffraction (Cu Kα) analyses (Figure 2, CCDC deposition number 1820072), recrystallized in CH2Cl2/MeOH (1:1).

Table 1. 1H (400 MHz) and 13C NMR (100 MHz) Data of 1 and 2 in DMSO-d6 1 No. 1-NH 2 3 4 5 6 7 8 9 10α 10β 11 12 13 14 16 17 18 19 20 21 22 23 24

δH (J in Hz)

2 δC

11.08, s

6.72, d (7.1) 6.96, dd (7.7,7.1) 7.16, d (7.7)

2.79, dd (15.1,11.0) 3.37, dd (15.1,5.4) 4.36, dd (11.0,5.4)

5.26, q (6.7) 1.52, d (6.7) 2.43, s 3.60, d (6.7) 5.27, t (6.7) 1.69, s 1.71, s

133.8 104.5 133.3 118.9 121.4 109.3 136.5 124.5 24.9 57.6 194.4a 102.0a 169.8a 43.1 20.2 183.0a 19.2 31.7 124.1 131.0 25.5 17.9

δH (J in Hz) 10.93, s 7.13, s

6.78, d (7.3) 6.96, dd (7.9,7.3) 7.17, d (7.9)

3.05, dd (15.8,5.0) 3.35, dd (15.8,6.3) 4.10, br s

6.27, d (8.5) 2.24, s 5.43, d (8.5) 1.68, s 1.83, s

δC 122.6 109.9b 136.5 116.8 120.9 109.9b 136.9 124.6 29.0 62.4a 194.2a 101.3a 172.1a 52.5 186.0a 22.9 123.9 134.9 25.3 18.4

a

Chemical shifts are read from a heteronuclear multiple bond correlation (HMBC) spectrum. bOverlapped.

(1H, d, 15.1, 5.4 Hz, H-10β), and 2.79 (1H, d, 15.1, 11.0 Hz, H-10α), one methine [δH 5.26 (1H, q, 6.7 Hz, H-16)] and an olefinic proton resonating at δH 5.27 (1H, t, 6.7 Hz, H-21). More structural details were derived from two-dimensional (2D) NMR spectral analyses. The correlation spectroscopy (COSY) cross signals between H-5/H-6 and H-6/H-7 of the spin system (−CHCH−CH) and heteronuclear multiple bond correlation (HMBC) correlations from H-5 to C-9, H-6 to C-4/C-8, H-7 to C-9, and 1-NH to C-2/C-8/C-9/C-16 demonstrated the presence of the indole part. At the meantime, the tetramic unit was supported on the basis of key HMBC correlations from H-11 to C-12/C-14 and 19-CH3 to C-13/C18 (see Figure 1). Interestingly, observed correlations from H16 to C-3/C-11/C-14, H-11 to C-3, and H-10 to C-3/C-9/C12 in HMBC spectrum suggested that the tetramic acid moiety was incorporated into the indole unit through two bridging carbons C-10 and C-16 to generate a six-membered Nheterocyclic ring C (Figure 1). As a result, a remarkable 6/5/6/ 5 tetracyclic ring system was identified to exist in structure 1. In

Figure 2. ORTEP structure of griseofamine A (1).

The absolute stereochemistry of 1 was elucidated by theoretical ECD calculation. Based on the relative configurations under the analyses of NOESY experiment and coupling constants, only two possible stereoisomers 1A (11S,16R) and 1B (11R,16S) of 1 existed. The experimental and simulated spectra generated by time-dependent density functional theory (TDDFT), which performed at B3LYP/631+G(d) level (Gaussian 16). As shown in Figure 3, the calculated ECD curve of 1A is in agreement with the experimental one. Therefore, the absolute configurations of C-11 and C-16 in 1 were assigned as 11S, 16R. The E

Figure 1. Key heteronuclear multiple bond correlation (HMBC), correlation spectroscopy (COSY), and nuclear Overhauser effect spectroscopy (NOESY) correlations of 1.

Figure 3. Comparison between calculated and experimental ECD spectra of 1 in MeOH. B


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double bond Δ13(17). As shown in Figure 5, calculated chemical shifts of C-12, C-13, and C-14 in 13Z-2 matched better with

configuration of the double bond Δ13(18) in 1 was also directly demonstrated from X-ray crystallography. Thus, compound 1 was deduced as a rare-type indole-tetramic alkaloid possessing a novel 6/5/6/5 tetracyclic ring system with a six-membered Nheterocyclic ring and named griseofamine A. Compound 2 was called griseofamine B and obtained as yellow oil. The positive-mode HRESIMS spectrum of 2 exhibited a [M + Na]+ ion peak at m/z 359.1361 that is consistent with a molecular formula of C20H20N2O3, requiring 12 degrees of unsaturation. The IR spectrum revealed the presence of a hydroxyl group at 3404 cm−1 and carbonyl groups at 1675 cm−1. The UV spectrum also showed two absorptions at 249 and 283 nm. Similar to that observed for 1, the resonances of 1D NMR and HSQC spectra (Table 1) recorded for 2 revealed the existence of the indole skeleton and the tetramic acid moiety that was further identified by key correlations of 1H−1H correlation spectroscopy (COSY) and HMBC spectra (see Figure 4).

Figure 5. Theoretical NMR calculation of two configurations of C-13 in 2.

the experimental data than the one with E configuration. Especially for C-14, the calculated value (δC 170.0) of 13Z-2 was much more similar to the experimental data rather than in 13E-2 (δC 163.1), which was consistent with above discussions. The relative configurations of two chiral centers (C-11 and C-16) in 2 were elucidated by the NOESY experiment. Key NOESY correlations between H-10β/H-11, H-11/H-19, H10β/H-19 and H-5/H-16 (Figure 6) indicated that H-10β and Figure 4. Key HMBC, COSY, and NOESY correlations of 2.

Nevertheless, the methyl assigned to C-17 in 1 was missing in the 1H NMR spectrum of 2, while a new olefinic proton singlet at δH 7.13 was attributed to C-2 of the indole moiety on the basis of COSY correlation between 1-NH and H-2 and key HMBC correlations from H-2 to C-3/C-8/C-9, from 1-NH to C-2 and from H-10 to C-2. In addition, a −CH−CH C(CH3)−CH3 group in 2 demonstrated that the methylene (20-CH2) of the isoprenyl chain in 1 was replaced by a methine at δH 6.27 (d, J = 8.5 Hz, H-16) and δC 52.5 (C-16), suggesting that compound 2 was an 3,4-disubstituted indole alkaloid possessing a new backbone rather than 1 with a 2,3,4trisubstituted indole alkaloid with a 6/5/6/5 tetracyclic ring system. Surprisingly, key HMBC correlations from H-10 to C2/C-9/C-11/C-12 and H-16 to C-5/C-9/C-11/C-14 (Figure 4) inferred the presence of a rare N-containing sevenmembered ring, which fused the tetramic acid scaffold on the indole skeleton via C-10 and C-16. Accordingly, the planar structure of 2 was deduced as shown. Generally, the two tautomers for the olefinic group in tetramic acid exist as Z-exo-enol and E-exo-enol form in a ratio that can occasionally be discriminated in 1H and 13C NMR spectra. In our case, the signals of the other tautomer have not been observed by spectroscopic analysis. The Z-exo-enol form with the hydroxy group hydrogen-bonded to the lactam carbonyl group is predominantly presented, which was elucidated by the lower field chemical shift than the corresponding normal carbonyl carbon.7−9 As a result, the chemical shift of C-14 (δ 172.1) in 2 designated the Δ13(17) Z stereochemistry for the tetramic acid.2 In addition, theoretical calculation of NMR chemical shifts, performed at the B3LYP/ 6-31G(d,p) level (Gaussian 09) in MeOH with PCM model, was applied for the determination of the configuration of the

Figure 6. Comparison between calculated and experimental ECD spectra of 2 in MeOH.

H-11 were on the same side of the seven-membered ring, while H-10α and H-16 were on the opposite side. The absolute stereochemistry of 2 was also merely two possibilities: 2A for 11S,16R and 2B for 11R,16S. To determine the absolute configuration of 2, ECD calculation was undertaken, allowing experimental and simulated spectra generated by TDDFT being compared. As shown in Figure 6, the experimental ECD data are basic anastomotic with the calculated ECD curve of 2A that assigned the absolute configurations of C-11 and C-16 as 11S, 16R. Therefore, compound 2 was determined as an indoletetramic acid alkaloid characterized an unusual 6/5/7/5 tetracyclic ring system featuring a seven-membered Nheterocyclic ring. Compound 3 was also isolated as a yellow oil and had the molecular formula C16H20N2O3 (8 degrees of unsaturation), as demonstrated from the positive-mode HRESIMS spectrum with a [M + H]+ ion peak at m/z 289.1562. The IR, UV spectrum, 1D NMR, and 2D NMR data indicated that compound 3 was also an indole-type alkaloid. The proton signals at δH 7.56 (d, J = 7.8 Hz, H-4), 7.32 (d, J = 7.6 Hz, H-7), C


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Figure 7. DP4 probabilities of diastereoisomers of 3.

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Scheme 1. Proposed Biosynthetic Pathway of 1−3

comprising unprecedented 6/5/6/5 and 6/5/7/5 tetracyclic ring systems, respectively, are isolated from P. griseof ulvum. Their structures are identified by NMR spectroscopic, theoretical NMR and ECD calculation, and X-ray diffraction. The activity screening of all compounds shows that only compound 1 exhibits a weak anti-inflammatory effect on the inhibition of the production of NO and TNF-α.

C NMR data for both

curve (Figure S2 in the Supporting Information), the absolute configuration of 3 was assigned as 11S, 15R. Thus, the structure of 3 was determined and given the name (R)-N-(2methylbutanoyl)-L-tryptophan. Based on the reported compound β-cyclopiazonic acid biosynthesized by a hybrid PKS-NRPS enzyme CpaS and a CpaD enzyme for the C-4 prenylation,3,4,10,11 the biosynthetic hypothesis of new structures 1−3 can be proposed as shown in Scheme 1. L-Tryptophan is also widely used as a building block for the biosynthesis of indole alkaloids. The premier intermediate (a) is assembled via a PKS-NRPS route from Ltryptophan and two molecules acetic acid, and then releases cyclo-acetoacetyl-L-tryptophan (cAATrp) catalyzed by a Dieckmann cyclase. The regioselective prenylation of indole moiety and further cyclization lead to the production of 2. To afford compound 1, a Mannich-like reaction, using pyruvic acid, and decarboxylation are considered to occur on 15-NH for the formation of ring C. For 3, it is successively generated through a keto reduction, dehydration, and enoyl reduction after an S-adenosylmethionine (SAM)-derived α-methylation from the intermediate (a). In the bioassay, compounds 1−3 were evaluated for antiinflammatory activity by inhibiting the production of NO and tumor necrosis factor-α (TNF-α) in the lipopolysaccharide (LPS)-induced RAW264.7 cells. Among them, compound 1 showed inhibitory activity against NO production with inhibition rate of 45.4% ± 2.8% at 10.0 μM (indomethacin was used as the positive control with the rate of 92.7% ± 2.5% at 50 μM) and the production of pro-inflammatory cytokine TNF-α with an IC50 value at 45.6 μM (dexamethasone was used as the positive control, IC50 15.2 μM). In conclusion, griseofamine A (1) and griseofamine B (2), which are two novel hybrid indole-tetramic acid alkaloids

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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.orglett.8b00584. Experimental details and copies of 1D and 2D NMR spectra and ECD calculations are disclosed; experimental details for X-ray analysis of 1 (PDF) Accession Codes

CCDC 1820072 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_ [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K.; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J. Wang). *E-mail: [email protected] (H. Zhu). *E-mail: [email protected] (Y. Zhang). ORCID

Grégory Genta-Jouve: 0000-0002-9239-4371 Yonghui Zhang: 0000-0002-7222-2142 Notes

The authors declare no competing financial interest. D


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ACKNOWLEDGMENTS This work was financially supported by the Program for Changjiang Scholars of Ministry of Education of the People’s Republic of China (No. T2016088), National Natural Science Foundation for Distinguished Young Scholars (No. 81725021), Innovative Research Groups of the National Natural Science Foundation of China (No. 81721005); the Academic Frontier Youth Team of HUST; the Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College (HUST); the Programe from the China Postdoctoral Science Foundation (No. 2017M622460). We thank the Analytical and Testing Center at Huazhong University of Science and Technology for assistance in testing of ECD, UV, and IR analyses.

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REFERENCES

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