Total Synthesis and Antibacterial Activity Evaluation of Griseofamine A

Mar 19, 2019 - Notably, 16-epi-griseofamine A was 2–3 times more potent than griseofamine A with MIC values of 2–8 μg/mL. The Supporting Informat...
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Total Synthesis and Antibacterial Activity Evaluation of Griseofamine A and 16-epi-Griseofamine A Xuan Pan and Zhanzhu Liu* State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, P. R. China

Org. Lett. Downloaded from pubs.acs.org by TULANE UNIV on 03/19/19. For personal use only.

S Supporting Information *

ABSTRACT: The first total synthesis of griseofamine A and its diastereomer, 16-epi-griseofamine A, is described over seven steps with yields of 23% and 7%, respectively. Their antibacterial activities are also disclosed for the first time. Griseofamine A exhibited in vitro activities against a panel of drug-resistant Gram-positive bacteria with minimum inhibitory concentration (MIC) values of 8−16 μg/mL. Notably, 16-epi-griseofamine A was 2−3 times more potent than griseofamine A with MIC values of 2−8 μg/mL.

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riseofamine A, a fungal secondary metabolite, was isolated by Zhang and co-workers in 2018 from the fungus Penicillium griseof ulvum.1 Structurally, it is characterized by an indole unit and a 3-acyl tetramic acid unit fused to a sixmembered nitrogen-containing heterocycle, making its core a totally new and unprecedented 6/5/6/5 tetracyclic ring system (Figure 1).

intriguing fragment, as this motif has been shown to improve the interactions between small molecules and proteins.18 The less complex structure and modifiability of griseofamine A, a unique indole-tetramic acid alkaloid, make it suitable as a lead compound for new drug discovery. For that reason, we decided to explore its chemical synthesis and biological properties. Herein, we report the first total synthesis of griseofamine A and its diastereomer, 16-epi-griseofamine A, from commercially available L-4-bromo tryptophan methyl ester in seven steps with overall yields of 23% and 7%, respectively. The antibacterial activities of griseofamine A and 16-epi-griseofamine A were also evaluated for the first time. The retrosynthetic analysis of griseofamine A is depicted in Scheme 1. The tetramic acid core of griseofamine A could be

Figure 1. Structure of griseofamine A.

Scheme 1. Retrosynthetic Analysis of Griseofamine A

Indoles are ubiquitous in natural products and pharmaceutically important molecules, and indole derivatives possess a wide range of biological activities.2−8 Moreover, indole scaffolds are well-known to interfere with numerous receptors with high affinity.9 Thus, the indole motif is considered one of the most privileged structures in medicinal chemistry. Tetramic acid (pyrrolidine-2,4-dione) units are common in the secondary metabolites of microorganisms and frequently have an acyl substituent at position 3 to afford 3-acyl tetramic acid. Natural products or bioactive molecules containing the 3acyl tetramic acid motif exhibit a variety of biological activities, including antibacterial, antiviral, and anticancer activities.10−13 Among these activities, antibacterial activity is of particular interest because 3-acyl tetramic acid was recently reported to play a crucial role in the quorum sensing of Gram-negative bacteria.14−17 The isoprenyl group of griseofamine A is also an © XXXX American Chemical Society

Received: February 22, 2019

A

DOI: 10.1021/acs.orglett.9b00672 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

in 92% yield. Using Buchwald’s protocol, the Suzuki−Miyaura cross-coupling reaction of compound 5a with 3,3-dimethyl allylboronate exclusively afforded linear prenylated product 6a in 80% yield, and none of the γ-prenylated product was observed. A few methods were used to remove the PMB group from compound 6a. CAN proved to be the only effective reagent for this transformation, but the loading of CAN must be carefully controlled. Addition of 1.2 equiv of CAN seemed to be most suitable for this reaction, even if an incomplete conversion was observed with the recovery of approximately 30% of 6a. Increasing the amount of CAN caused decomposition of starting material 6a and product 7a. When the amount of CAN was increased to 4 equiv, 6a and 7a were completely degraded. Recovered 6a could again be subjected to the reaction, which eventually resulted in a 70% yield of compound 7a. Boc deprotection of compound 7a was then conducted. It is well-known that carbon−carbon double bonds are sensitive to acids. Indeed, we obtained the corresponding adducts when compound 7a was exposed to TFA or HCl. Attempts to achieve Boc deprotection by decreasing the reaction temperature or the concentration of acid failed to give any positive results. Basic conditions (CH3ONa/CH3OH/ THF), a common strategy for the Boc deprotection of indole nitrogens, were also unsuccessful in our study. Hydrolysis of the ester was observed, while the desired product was not. Finally, neutral conditions (TMSOTf and 2,6-lutidine) proved to be effective in our experiment, and they enabled the successful removal of the Boc group in 90% yield. N-Acylation of 8a with an acetyl ketene was carried out in the presence of Et3N in DCM at room temperature. Gratifyingly, compound 8a was directly converted to the final product, griseofamine A, in excellent yield (87%) (Scheme 3). The tandem acylation/

accomplished via a base-promoted Lacey−Dieckmann condensation of β-keto amide B. β-Keto amide intermediate B could be generated by the reaction of 1,2,3,4-tetrahydro-βcarboline C with an acetyl ketene. Selective prenylation of intermediate D could be achieved through the Suzuki− Miyaura cross-coupling reaction described by Buchwald, which would allow the highly α-selective coupling of 3,3dimethyl allylboronate with an aryl bromide to afford a linear prenylated product with excellent regioselectivity.19 trans1,2,3,4-Tetrahydro-β-carboline D would be readily prepared via the Pictet−Spengler cyclization established by Cook and co-workers in 1979, allowing the smooth conversion of Nbprotected tryptophan methyl ester to the thermodynamically more stable trans-1,2,3,4-tetrahydro-β-carboline with high diastereoselectivity.20−24 An appropriate modification was made in our study; that is, a p-methoxybenzyl group (PMB) was used instead of the benzyl group used in Cook’s method due to its tolerance of various harsh conditions and facile removal by cerium ammonium nitrate (CAN). Finally, intermediate E could be obtained from L-4-bromo tryptophan methyl ester and p-anisaldehyde via a reductive amination. Our study commenced with the introduction of the PMB group to commercially available L-4-bromo tryptophan methyl ester (2) via a reductive amination (Scheme 2). Treatment of Scheme 2. Synthesis of Key Intermediates 1,2,3,4Tetrahydro-β-carboline 4a and 4b

Scheme 3. Synthesis of Griseofamine A and 16-epiGriseofamine A

L-4-bromo tryptophan methyl ester (2) with p-anisaldehyde in the presence of MgSO4 in C2H5OH followed by the addition of NaBH4 generated compound 3 in 82% yield. As anticipated, the stereoselective Pictet−Spengler cyclization of compound 3 with acetaldehyde in refluxing CH2Cl2 predominantly yielded desired trans-1,2,3,4-tetrahydro-β-carboline 4a (69%) along with cis-1,2,3,4-tetrahydro-β-carboline 4b (22%) as a minor product in a ratio of approximately 3:1. The diastereomers were conveniently separated by column chromatography. Their configurations were confirmed by a NOESY experiment, which showed an obvious NOESY correlation between H-1 and H-3, suggesting a cis configuration in compound 4b. In contrast, no correlation between H-1 and H-3 was observed in compound 4a, indicating a trans configuration. Additionally, cis isomer 4b could be converted to the thermodynamically more stable trans isomer (4a) in TFA via the cleavage and reformation of the C(1)−N(2) bond in 85% yield, which improved the total yield of trans-4a to 88%. Certainly, cis1,2,3,4-tetrahydro-β-carboline 4b could serve as the key intermediate in the synthesis of 16-epi-griseofamine A. Protection of the indole nitrogen of 4a with Boc2O in the presence of Et3N and DMAP in DCM furnished compound 5a

Lacey−Dieckmann condensation avoided the use of strong bases (CH3ONa, tBuOK, and NaH), which are commonly used in Lacey−Dieckmann cyclizations. Griseofamine A was finally obtained as a white solid in 23% yield over seven steps. If the conversion of cis-1,2,3,4-tetrahydro-β-carboline 4b into trans-1,2,3,4-tetrahydro-β-carboline 4a was taken into account, the total yield of griseofamine A was 29%. Its 1H and 13C NMR spectra were fully consistent with the natural product reported B

DOI: 10.1021/acs.orglett.9b00672 Org. Lett. XXXX, XXX, XXX−XXX

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Table 1. Antibacterial Evaluation of Griseofamine A and 16-epi-Griseofamine A (MIC values in micrograms per milliliter)a entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

strain c

S. aureus (ATCC29213) S. aureus (15)d S. aureus (16-1)d MRSA (ATCC33591)c MRSA (16-30)d S. epidermidis (ATCC12228)c S. epidermidis (16-4)d MRSE (16-5)d E. faecalis (ATCC29212)c E. faecalis (16-6)d VRE (ATCC51299)c,e VRE (ATCC51575)c,e E. faecium (16-5)d VRE (ATCC700221)c,f VRE (12-1)d,f Escherichia coli (ATCC25922)c Pseudomonas aeruginosa (ATCC 27853)c Klebsiella pneumoniae (ATCC 700603)c Acinetobacter baumannii (ATCC19606)c

griseofamine A

16-epi-griseofamine A

CIPb

8 8 8 8 8 8 8 8 16 16 16 16 16 16 16 >128 >128 >128 >128

2 2 2 2 4 2 2 4 8 4 4 8 8 8 8 >128 >128 >128 >128

0.25 0.25 0.25 0.25 128 0.12 0.12 32 0.5 1 0.5 0.5 64 128 64 ≤0.03 0.5 0.25 0.5

a

MIC values were determined by a standard serial 2-fold dilution method. bCiprofloxacin (CIP) was used as the reference. cATCC standard strains. Representative clinical isolates from China. eVancomycin-resistant E. faecalis. fVancomycin-resistant E. faecium.

d

of 2−8 μg/mL, suggesting the crucial role of the stereochemistry in the antibacterial activity. In most cases, the activities of griseofamine A and 16-epi-griseofamine A were slightly weaker than that of CIP. However, in the cases of MRSA (16-30) and MRSE (16-5), both compounds exerted antibacterial activities significantly higher than that of CIP (entries 5 and 8, respectively). In addition, E. faecium and vancomycin-resistant E. faecium were not sensitive to CIP (MIC values of 64 and 128 μg/mL, respectively), whereas our compounds exhibited potential inhibitory activity toward these strains with MIC values of 16 μg/mL for griseofamine A and 8 μg/mL for 16-epi-griseofamine A (entries 13−15). In conclusion, we have completed the first total synthesis of griseofamine A and its diastereomer, 16-epi-griseofamine A, on a gram scale from L-4-bromo tryptophan methyl ester over seven steps in 23% and 7% overall yields, respectively. If the conversion of cis-1,2,3,4-tetrahydro-β-carboline 4b to trans1,2,3,4-tetrahydro-β-carboline 4a is considered, the total yield of griseofamine A is 29%. The yield of 16-epi-griseofamine A was relatively low (7%) because cis-1,2,3,4-tetrahydro-βcarboline 4b was not the major product of the Pictet− Spengler reaction described by Cook. However, the cis-1,2,3,4tetrahydro-β-carboline could be easily obtained through a conventional Pictet−Spengler reaction, which would significantly enhance the overall yield of 16-epi-griseofamine A. The whole procedure was scalable and featured three key transformations: (1) the diastereoselective construction of the trans-1,2,3,4-tetrahydro-β-carboline from Nb-PMB tryptophan methyl ester and an acetaldehyde via a Pictet−Spengler reaction, (2) the introduction of an isoprenyl group by a Suzuki−Miyaura cross-coupling reaction to afford the linear prenylated product in a highly regioselective manner, and (3) the preparation of the 3-acyl tetramic acid moiety via a tandem N-acylation/Lacey−Dieckmann condensation. Griseofamine A was also found to exhibit potent antibacterial activity against various drug-resistant Gram-positive bacteria (MRSA, MRSE, and VRE) with MIC values of 8−16 μg/mL for the first time. Moreover, 16-epi-griseofamine A was more active than the

by Zhang.1 Likewise, only the Z-exoenol tautomer was observed. Accordingly, 16-epi-griseofamine A was prepared from intermediate 4b in 39% yield over five steps. Because cis1,2,3,4-tetrahydro-β-carboline 4b was the minor product of the Pictet−Spengler reaction and was obtained in a yield of 22%, the total yield of 16-epi-griseofamine A was only 7% from the starting material (L-4-bromo tryptophan methyl ester). Clearly, 16-epi-griseofamine A could be prepared through other synthetic routes in good yields. The structure of 16-epigriseofamine A was confirmed by 1H and 13C NMR spectroscopy and HRMS. With these two compounds in hand, we then sought to preliminarily investigate their antibacterial activities against a panel of Gram-positive and Gram-negative bacteria. These strains included standard bacteria obtained from the American Type Culture Collection (ATCC) and representative clinical isolates from China. Methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), and vancomycin-resistant Enterococcus (VRE), representing the most common drug-resistant pathogens in clinical and public settings, were also examined. The minimum inhibitory concentrations (MIC values) were determined by a standard serial 2-fold dilution method. Ciprofloxacin (CIP), a standard antibiotic, was used as the positive control. The antibacterial activities are listed in Table 1. As shown in Table 1, griseofamine A and its diastereomer, 16-epi-griseofamine A, exhibited specific inhibitory activities against Gram-positive bacteria, including various drug-resistant bacteria. Comparable efficiencies were observed between drugsensitive pathogens and the corresponding drug-resistant pathogens. For instance, griseofamine A was effective against both S. aureus and MRSA with MIC values of 8 μg/mL (entries 1−5). Griseofamine A also inhibited the growth of both S. epidermidis and MRSE with MIC values of 8 μg/mL (entries 6−8). It also inhibited Enterococcus faecalis, Enterococcus faecium, and VRE with MIC values of 16 μg/mL (entries 9−15). 16-epi-Griseofamine A was 2−3-fold more potent than its diastereomer griseofamine A with MIC values C

DOI: 10.1021/acs.orglett.9b00672 Org. Lett. XXXX, XXX, XXX−XXX

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(14) Han, J.; Liu, C.; Li, L.; Zhou, H.; Liu, L.; Bao, L.; Chen, Q.; Song, F.; Zhang, L.; Li, E.; Liu, L.; Pei, Y.; Jin, C.; Xue, Y.; Yin, W.; Ma, Y.; Liu, H. J. Org. Chem. 2017, 82, 11474−11486. (15) Hofmann, L. E.; Mach, L.; Heinrich, M. R. J. Org. Chem. 2018, 83, 431−436. (16) Josa-Cullere, L.; Pretsch, A.; Pretsch, D.; Moloney, M. G. J. Org. Chem. 2018, 83, 10303−10317. (17) Kong, L.; Rao, M.; Ou, J.; Yin, J.; Lu, W.; Liu, M.; Pang, X.; Gao, S. Org. Biomol. Chem. 2014, 12, 7591−7597. (18) Backhaus, K.; Ludwig-Radtke, L.; Xie, X.; Li, S. M. ACS Synth. Biol. 2017, 6, 1056−1064. (19) Yang, Y.; Buchwald, S. L. J. Am. Chem. Soc. 2013, 135, 10642− 10645. (20) Stockigt, J.; Antonchick, A. P.; Wu, F.; Waldmann, H. Angew. Chem., Int. Ed. 2011, 50, 8538−8564. (21) Ungemach, F.; DiPierro, M.; Weber, R.; Cook, J. M. Tetrahedron Lett. 1979, 20, 3225−3228. (22) Zhang, L. H.; Cook, J. M. Heterocycles 1988, 27, 1357−1363. (23) Ungemach, F.; DiPierro, M.; Weber, R.; Cook, J. M. J. Org. Chem. 1981, 46, 164−168. (24) Cox, E. D.; Hamaker, L. K.; Li, J.; Yu, P.; Czerwinski, K. M.; Deng, L.; Bennett, D. W.; Cook, J. M.; Watson, W. H.; Krawiec, M. J. Org. Chem. 1997, 62, 44−61.

natural product (griseofamine A) against drug-resistant bacteria with MIC values of 2−8 μg/mL. These results indicate that griseofamine A is a potential lead compound for the development of novel antibacterial agents to combat drugresistant bacteria. Systematic structural modifications of griseofamine A are ongoing in our laboratory, and the results will be reported in due course.

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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.9b00672.

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Experimental procedures and characterization data of new compounds (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Zhanzhu Liu: 0000-0002-2181-5626 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, the CAMS Innovation Fund for Medical Sciences (CIFMS, 2016I2M-3-009), the nonprofit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018PT35002), and the Drug Innovation Major Project (2018ZX09711-001005-014 and 2018ZX09711-001-005).

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DOI: 10.1021/acs.orglett.9b00672 Org. Lett. XXXX, XXX, XXX−XXX