Polycyclic Macrolactams Generated via Intramolecular Diels–Alder


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

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Polycyclic Macrolactams Generated via Intramolecular Diels−Alder Reactions from an Antarctic Streptomyces Species Jingjing Shen,†,∥ Yaqin Fan,†,∥ Guoliang Zhu,† Hao Chen,§ Weiming Zhu,*,†,‡ and Peng Fu*,†,‡ †

Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China ‡ Laboratory for Marine Drugs and Bioproducts, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266003, China § Key Laboratory of Marine Bioactive Substances, First Institute of Oceanography, Ministry of Natural Resources of China, Qingdao 266061, China

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S Supporting Information *

ABSTRACT: Three new polycyclic macrolactams, cyclamenols B−D (1−3), together with a known macrolactam, cyclamenol A (4), were isolated from the Streptomyces sp. OUCMDZ-4348. Their structures including absolute configurations were determined on the basis of spectroscopic analysis, chemical methods, and ECD calculations. The biosynthetic pathways involving intramolecular Diels−Alder reactions were proposed. Compound 1 exhibited selective inhibition against the gastric carcinoma cell line N87 with an IC50 value of 10.8 μM.

T

metabolites of OUCMDZ-4348 and identified three new polycyclic macrolactams, cyclamenols B−D (1−3), along with the known cyclamenol A (4).

he Diels−Alder reaction is one of the most powerful chemical reactions in the synthesis of small molecules, which is usually used to form a complex carbocyclic system. This reaction could be carried out under mild conditions with high regio- and stereoselectivity. Thus, it has become a useful tool for forming chiral centers in the synthesis of biological active compounds.1 In recent years, many natural products formed via an intramolecular Diels−Alder route have been identified, and some enzymes that can catalyze Diels−Alder reactions have been discovered.2 The biosynthetic pathways of some microbial natural Diels−Alder-type cycloadducts have been elucidated, such as varicidins,3 spinosyns,4 leporins,5 and abyssomicin C.6 Polyene macrolactams are a large family of natural products, which are usually found in actinomycetes.7 The structural characteristics of polyene macrolactams make them important precursors of natural Diels−Alder reactions. Some Diels−Alder-type cycloadducts derived from macrolactams have been isolated from actinomycetes, such as verticilactam,8 macrotermycins B and D,9 and clifednamides A and B.10 Our research has focused on the active natural products of marine-derived actinomycetes. We have discovered some bioactive compounds with novel structures, such as cyanogrisides,11 streptocarbazoles,12 cyanogramide,13 and fradcarbazoles.14 As part of our ongoing studies to search for novel active molecules from actinomycetes, the Streptomyces sp. OUCMDZ-4348 (GenBank accession no. MK634671) was isolated from a sand sample collected from the Antarctic. LC− MS and 1H analyses showed this strain could produce macrolactams. Considering the structural diversity and biological activity of macrolactams, we investigated the secondary © XXXX American Chemical Society

Cyclamenol B (1) was obtained as a white powder. Its molecular formula was determined as C20H25NO3 based on the Received: May 15, 2019

A

DOI: 10.1021/acs.orglett.9b01710 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters HRESIMS peak at m/z 328.1908 [M + H]+ (Figure S4). The 13 C NMR spectrum (Figures S7 and S8) showed 20 signals which were classified by HSQC (Figure S9) as one carbonyl, eight sp2-methine carbons, eight sp3-methine carbons (three oxygenated ones), two methylene carbons, and one methyl carbon. The 1H NMR spectrum (Figures S5 and S6) showed one methyl signal at δH 0.96 (d, J = 6.7 Hz) and eight olefin signals at δH 4.98 (“t” like, J = 10.0 Hz), 5.51 (dd, J = 15.4, 8.6 Hz), 5.58 (d, J = 9.5 Hz), 5.80 (d, J = 15.6 Hz), 5.80 (overlapped), 5.84 (overlapped), 5.88 (dd, J = 15.5, 9.9 Hz), and 6.15 (dd, J = 15.3, 11.2 Hz). There was also a exchangeable proton at δH 7.61 (d, J = 10.0 Hz), which could be classified as an amide by the HMBC correlation of NH to C-1 (δC 164.6) (Figure 1). The contiguous COSY

B) and a cyclopentane (ring C), respectively. The connection among these three rings was determined by the COSY correlations of H-7/H-11 and H-4/H-12 (Figure 1). C-9 (δC 72.4), C-10 (δC 80.1), and C-13 (δC 82.7) (Table 1) were oxygenated carbons. The HMBC correlation of H-10 to C-13 indicated the presence of a tetrahydrofuran ring (ring D). Thus, the planar structure of compound 1 could be elucidated as shown. The configurations of double bonds at Δ2, Δ14, and Δ16 were assigned as E, E, and Z by the coupling constants of 15.6, 15.4, and 10.0 Hz, respectively. The relative configuration of compound 1 was assigned as (4R*,7R*,9R*,10S*,11R*,12R*,13S*) by the NOESY correlations of H-4/H-13, H-4/H-11, H-3/H-12, H-11/H-13, H-10/H-11, H-8a/H-12, H-8b/H-7, H-8b/H-9, H-7/H-9, H-9/H-10, and H-12/H-14 (Figure 2).

Figure 1. Key 2D NMR correlations of compounds 1−3.

correlations extending from H-12 to NH and the correlations of H-2/H-3/H-4/H-12, together with the HMBC correlations of H-2/H-3/H-19 to C-1 (Figure 1), indicated the presence of a 13-membered lactam ring (ring A). Meanwhile, the COSY correlations of H-4/H-5/H-6/H-7/H-11/H-12 and H-7/H28/H-9/H-10/H-11 (Figure 1) suggested a cyclohexene (ring

Figure 2. Key NOESY correlations of compounds 1 and 2.

The molecular formula of cyclamenol C (2) was also determined to be C20H25NO3 by HRESIMS, which is an isomer of 1. Comparison of its 1H and 13C NMR spectra

Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for Cyclamenols B and C (1 and 2) in DMSO-d6 1 no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 -NH −OH

δC 164.6, 128.4, 141.4, 43.9, 130.2, 131.8, 34.4, 39.4, 72.4, 80.1, 50.9, 49.7, 82.7, 135.6, 128.1, 129.4, 134.2, 34.4, 44.8, 18.9,

C CH CH CH CH CH CH CH2 CH CH CH CH CH CH CH CH CH CH CH2 CH3

2 δH, mult (J in Hz)

5.80, 5.88, 3.02, 5.58, 5.84, 2.40, 1.44, 3.94, 4.18, 2.40, 1.60, 4.06, 5.51, 6.15, 5.80, 4.98, 2.40, 2.73, 0.96, 7.61,

δC

d (15.6) dd (15.5, 9.9) “t” like (10.1) d (9.5) overlapped overlapped m; 2.24, m “q” like (7.5) “t” like (6.9) overlapped “q” like (10.8) “t” like (9.5) dd (15.4, 8.6) dd (15.3, 11.2) overlapped “t” like (10.0) overlapped d (12.9); 3.25, “q” like (10.5) d (6.7) d (10.0)

B

164.6, 128.5, 141.3, 44.1, 130.6, 132.1, 38.3, 39.4, 78.8, 89.9, 50.9, 49.7, 82.6, 135.7, 128.4, 129.4, 134.3, 34.4, 44.8, 18.9,

C CH CH CH CH CH CH CH2 CH CH CH CH CH CH CH CH CH CH CH2 CH3

δH, mult (J in Hz) 5.80, 5.85, 3.02, 5.60, 5.85, 2.78, 1.75, 3.96, 4.10, 2.47, 1.39, 3.96, 5.26, 6.14, 5.80, 4.96, 2.40, 2.69, 0.95, 7.60, 4.77,

d (15.4) dd (15.4, 10.2) “t” like (10.3) d (9.7) overlapped brs dd (13.8, 8.1); 1.92, m overlapped d (7.2) “q” like (6.3) “q” like (11.0) “t” like (9.5) dd (15.3, 8.7) dd (15.2, 11.4) “t” like (10.0) “t” like (10.2) m d (12.7); 3.27, “q” like (11.3) d (6.7) d (9.6) brs DOI: 10.1021/acs.orglett.9b01710 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters (Table 1 and Figures S14−S17) with those of compound 1 revealed that their NMR data were very similar except for C-7, C-9, and C-10. Analysis of its 2D NMR correlations (Figure 1) suggested that compound 2 has the same planar structure as that of 1. Those differences of chemical shifts indicated that 2 might be an epimer of 1 at C-9, which was also confirmed by the NOESY correlations of H-4/H-13, H-4/H-11, H-3/H-12, H-11/H-13, H-10/H-11, H-8a/H-12, H-8a/H-9, H-8b/H-7, and H-12/H-14 (Figure 2). The molecular formula of cyclamenol D (3) was determined to be C22H27NO3 by HRESIMS. The 1H and 13C NMR data (Table S1), assigned by HSQC, COSY, and HMBC correlations (Figure 1), indicated the presence of two exchangeable protons at δH 7.98 (d, J = 9.4 Hz) and 4.63 (d, J = 5.2 Hz), two carbonyl groups (δC 163.8 and 190.9), five double bonds, six sp3-methine groups, three methylene groups, and one methyl group. Compound 3 also has an amide, which was determined by the key HMBC correlation of NH to C-1 (Figure 1). The COSY correlations of H-4/H-5/H-6/H-15/H16/H-17/H-18/H-19/H-20/H2-21/NH, and H-20/H3-22, together with the key HMBC correlations of H2-2 to C-1/C3, H-5 to C-3/C-4, NH to C-21, and H2-21 to C-1 (Figure 1), revealed the presence of a 14-membered lactam ring (ring A). The structures of two cyclohexene units (rings B and C) were determined by the COSY correlations of H-6/H-7/H-12/H13/H-14/H-15, H-7/H-8/H-9, and H-10/H-11/H-12. The key HMBC correlations of H-9 to C-8/C-10/C-11 (Figure 1) further support this structural moiety. The connection of these three rings was assigned by the COSY correlations of H-6/H15 and H-7/H-12. Further analysis of 2D NMR signals (Figure 1) gave the complete structural assignment of compound 3. The configurations of double bonds at Δ4, Δ16, and Δ18 were all assigned as E by the coupling constants of 15.5, 15.0, and 15.0 Hz. Its relative configuration was determined as (6S*,7R*,11S*,12S*,15R*) by the key NOESY correlations of H-5/H-7, H-5/H-15, H-6/H-16, H-6/H-11, H-10a/H-11, H-10b/H-12, H-10b/H-7, H-7/H-12, and H-7/H-15 (Figure 3).

Scheme 1. Chemical Reactions for the Identification of the Absolute Configuration at C-18 of 4

generate compound 5, which was further reacted with (S)- and (R)-phenylglycine methyl ester (PGME) to yield (S)- and (R)PGME amides (5a and 5b) of 5.16 The chemical shift differences ΔδS−R (Scheme 1) defined the R configuration of compound 5. Thus, the absolute configuration of C-18 in compound 4 was determined as S. The absolute configuration of C-9 in 4 was established as R by the modified Mosher’s method (Figure 4).17

Figure 4. Δδ (= δS − δR) values for (S)- and (R)-MTPA esters of compounds 1−4.

The absolute configurations of C-9 in 1 and 2 and C-11 in 3 were determined as R, R, and S by the modified Mosher’s method (Figure 4).17 In the process, we found that the Δδ value at H-10 for compound 1 was inconsistent. In order to explain this inconsistent value, DFT modeling for the MTPA esters of 1 was performed (Figure S58). The lowest energy conformations indicated that H-10 was deshielded by the phenyl ring in the (S)-MTPA ester, while it was not affected in the (R)-MTPA ester. The absolute configurations of the carbons connected with methyl group in compounds 1−3 (C18 in 1 and 2, C-20 in 3) were all determined by ozonolysis, acid hydrolysis, and derivatization. The products of ozonolysis

Figure 3. Key NOESY correlations of compound 3.

Cyclamenol A (4) has been reported in a patent. It was isolated from the Streptomyces strain MHW 846. This compound was found to have leukocyte adhesion inhibiting activity.15 However, the absolute configuration had not been determined. To determine the absolute configuration of C-18 of cyclamenol A (4), ozonolysis and acid hydrolysis were carried out (Scheme 1).7a,d The product 3-amino-2-methylpropanoic acid was then derivatized with Sanger’s reagent to C

DOI: 10.1021/acs.orglett.9b01710 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

isomeric intermediates (b and c). These intermediates further underwent dehydration to form the tetrahydrofuran ring of cyclamenols B and C (1 and 2). Cyclamenol D (3) was also formed via a Diels−Alder reaction from a 22-membered macrolactam (d). Compounds 1−4 were evaluated for cytotoxicity by the Cell Titer Glo (CTG) assay.20 In this assay, 26 human cancer cell lines and two normal ones (the names of cell lines could be found in the Supporting Information). The results showed that only compound 1 exhibited moderate selective activity against the gastric carcinoma cell line N87 with an IC50 value of 10.8 μM. No cytotoxic activity against other cell lines was observed, which indicated that compound 1 might be acting on a specific target of N87 cell line. In summary, we identified three new polycyclic macrolactams (1−3) from an Antarctic Streptomyces strain. These compounds were derived from macrolactams via intramolecular Diels−Alder reactions. The formation mechanism of these natural products could provide objects for the study of Diels−Alder reactions in microbes. Meanwhile, the selective cytotoxic effect of compound 1 gave a direction for further investigation of anticancer activity.

and hydrolysis for compounds 1−3 were treated with (S)PGME to yield (S)-PGME amides, which were identified to be same as 5a by comparison of their retention times on HPLC with 5a and 5b derived from compound 4 (Figure S3). Thus, the absolute configurations of compounds 1−3 were clearly determined as (4R,7R,9R,10S,11R,12R,13S,18S), (4S,7S,9R,10R,11S,12S,13R,18S), and (6S,7R,11S,12S,15R,20S), respectively. Furthermore, the calculated ECD spectrum of 1 was obtained by the TDDFT [B3LYP/6-31G(d)] method,18 which matched the experimental data (Figure 5).



ASSOCIATED CONTENT

S Supporting Information *

Figure 5. Experimental and calculated ECD curves for compound 1.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b01710.

The plausible biosynthetic pathways for compounds 1−4 were postulated (Scheme 2). Glutamic acid and acetyl-CoA19 were utilized to form the 20-membered macrolactam, cyclamenol A (4), which underwent epoxidation, hydrolysis, elimination, and isomerization of double bond to yield the intermediate a. This important intermediate underwent potential Diels−Alder cycloaddition to yield two stereo-



Scheme 2. Postulated Biosynthesis of Compounds 1−4

Experimental details, ECD curves, NMR table for compounds 3 and 4, HRESIMS and NMR spectra, and details for calculations (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] *E-mail: [email protected] ORCID

Weiming Zhu: 0000-0002-7591-3264 Peng Fu: 0000-0002-7768-4004 Author Contributions ∥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 41806086 and 41876172), the National Key R&D Program of China (No. 2018YFC1406705), and the Fundamental Research Funds for the Central Universities (No. 201841006). We thank Ming Li and Peng Wang (School of Medicine and Pharmacy, Ocean University of China, Qingdao, China) for guidance with the chemical reactions.



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DOI: 10.1021/acs.orglett.9b01710 Org. Lett. XXXX, XXX, XXX−XXX