Ten-step asymmetric total syntheses of potent antibiotics anthracimycin and anthracimycin B

The increase in antibiotic resistance calls for the development of novel antibiotics with new molecular structures and new modes of action. However, in the past few decades only a few novel antibiotics have been discovered and progressed into clinically used drugs. The discovery of a potent anthracimycin antibiotic represents a major advance in the field of antibiotics. Anthracimycin is a structurally novel macrolide natural product with an excellent biological activity profile: (i) potent in vitro antibacterial activity (MIC 0.03–1.0 μg mL−1) against many methicillin-resistant Staphylococcus aureus (MRSA) strains, Bacillus anthracis (anthrax), and Mycobacterium tuberculosis; (ii) low toxicity to human cells (IC50 > 30 μM); (iii) a novel mechanism of action (inhibiting DNA/RNA synthesis). While the first total synthesis of anthracimycin was elegantly accomplished by Brimble et al. with 20 steps, we report a 10-step asymmetric total synthesis of anthracimycin and anthracimycin B (first total synthesis). Our convergent strategy features (i) one-pot sequential Mukaiyama vinylogous aldol/intramolecular Diels–Alder reaction to construct trans-decalin with high yield and excellent endo/exo selectivity and (ii) Z-selective ring-closing metathesis to forge the 14-membered ring. In vitro antibacterial evaluation suggested that our synthetic samples exhibited similar antibacterial potency to the naturally occurring anthracimycins against Gram-positive strains. Our short and reliable synthetic route provides a supply of anthracimycins for further in-depth studies and allows medicinal chemists to prepare a library of analogues for establishing structure–activity relationships.


Introduction
The alarming prevalence of bacteria resistant to multiple antibiotics raises a global concern on the availability of new antibiotics effective against such "superbugs" that are resistant to most or all known antibiotics. 1,2 The search for new antibiotics with different strategies including bioassay-guided isolation of new antibacterial natural products, genome mining, [3][4][5][6][7][8] and chemical synthesis/modications of existing antibiotics 9,10 continues to play an important role in addressing antibiotic resistance. [11][12][13][14] However, only a few structurally novel antibacterial compounds have been discovered in the past ve decades, and anthracimycin 15 was one of these new antibiotics with a novel molecular structure and high antibacterial potency. Anthracimycin was discovered in 2013 by Fenical and co-worker from marine microorganism Streptomyces species (CNH365) to be a structurally novel potent antibiotic against Gram-positive bacteria (MIC 0.03-0.5 mg mL −1 ) including notorious methicillin-resistant Staphylococcus aureus (MRSA) 16 and the bacterium Bacillus anthracis that has been used as a bioterrorism weapon to cause human-infectious anthrax (Fig. 1). In 2018, Reyes et al. 17 identied 2-demethyl anthracimycin (anthracimycin B) from cultures of the deep-sea actinomycete Streptomyces cyaneofuscantus M-169 and found that it exhibited similar but less potent antibacterial activity against Gram-positive bacteria. Another anthramycin analogue (anthracimycin BII-2619) was also identied in 2018 by Sirota et al. 18 through a genome mining strategy to display Gram-positive antibacterial activity with lower mammalian cytotoxicity compared to that of anthracimycin. In 2020, Fukuda 19 and co-workers isolated 2-epianthracimycin along with anthracimycin and evaluated its cytotoxicity against Jurkat cells (IC 50 = 50.5 mM). While most macrolide antibiotics bind to the 50S ribosomal subunit to inhibit protein biosynthesis, 20 it was suggested that anthracimycin exerts its bactericidal effect through inhibiting DNA and/ or RNA synthesis in the absence of DNA intercalation. 21 The high antibacterial potency, new molecular structure, and novel mechanism of action make anthracimycin an ideal target for chemical synthesis and development of novel antibiotics.
In 2019, two PhD theses (J. L. Freeman 22,23 and G. Lodovici 24 ) disclosed detailed synthetic studies towards anthracimycin and presented a signicant synthetic challenge of using the biomimetic intramolecular Diels-Alder (IMDA) reaction of the tetraene substrate (Scheme 1b) 25,26 to construct the decalin core. Further optimization of Freeman's thesis work enabled Brimble 27 and co-workers to achieve the rst total synthesis of anthracimycin in 20 steps with 0.4 mg pure anthracimycin. It was noted that Kalesse et al. 28 accomplished the total synthesis of structurally related chlorotonil A 29 in 21 steps by using the halogen-directed IMDA reaction and macrolactonization as the key steps. In this paper, we report a 10-step asymmetric total synthesis of anthracimycin and anthracimycin B (the rst total synthesis) with substantially improved overall yield (>3% overall yield, 25 mg and 14 mg obtained, respectively).

Results and discussion
The key synthetic challenge of anthracimycin in the previous synthetic studies [22][23][24]27 was the inefficient construction of the trans-decalin core through the biomimetic intramolecular Diels-Alder (IMDA) reaction of the tetraene substrate. To address this challenge, we proposed an IMDA reaction of an "unusual" triene substrate VII with a C9 secondary alcohol (Scheme 1), which served (1) as the precursor of the trisubstituted Z-9,10 D alkene, (2) to enhance the endo/exo selectivity and diastereoselectivity, and (3) to improve the convergency of the synthesis because the presence of the alcohol would allow the fragment union through C-C bond formation with many wellestablished transformations (i.e., aldol, allylation, Grignard addition, etc.). With this central ideal in mind, our retrosynthetic analysis was conceived as depicted in Scheme 1. The 14-membered macrocycle would be forged in the last step by either macrolactonization 28 of I or Z-selective ring-closing metathesis (RCM) of II. Notably, Z-selective RCM might be advantageous since the preparation of the more challenging Z-congured alkene substrate at an early stage of synthesis was eliminated. Both I and II can be obtained by condensation of 1,3-dicarbonyl V with III and IV, respectively. The designed triene VII substrate for the IMDA reaction could be easily synthesized by Julia-Kocienski olenation 30 of aldehyde X and tetrazole XI followed by the Mukaiyama vinylogous aldol (MVA) 31-35 reaction of aldehyde VIII and O,O-silyl ketene acetal IX. 36 Our synthesis consisted of two stages: trans-decalin synthesis and macrolactone synthesis as depicted in Scheme 2. We started with the preparation of known compound 2 (ref. 37 and 38) in 86% yield by Evans asymmetric alkylation 39 of commercially available (S)-4-benzyl-3-propionyl-2-oxazolidinone (1). Olen cross metathesis of 2 with crotonaldehyde delivered 3 as an exclusive E-conguration in 85% yield. Julia-Kocienski olenation of N-phenyl tetrazole sulfone 4a (ref. 22 and 40) with aldehyde 3 followed by Dess-Martin periodinane (DMP) oxidation provided (E,E)-diene 5a (E/Z > 10 : 1), which was subjected to our planned Lewis acid-promoted Mukaiyama vinylogous aldol (MVA) reaction with the known silyl ketene acetal 6. 36,41 Triarylborane [B(C 6 F 5 ) 3 ] 42 was found to effectively promote MVA to provide triene 7 in 71% yield with a 6 : 1 diastereomeric ratio. The thermal IMDA reaction of triene 7 was rst attempted at 165°C in dichlorobenzene to produce a mixture of endo/exo products (endo/exo: 2.3 : 1) with 82% combined yield. To improve the endo/exo selectivity, we used BF 3 -Et 2 O to promote the IMDA reaction, 29 which resulted in decomposition. Fortunately, we found that Et 2 AlCl (4.0 equiv.) was an effective promoter for the IMDA reaction to provide the desired trans-decalin as a mixture of 8a and 8b, which upon treatment with 37% HCl for removal of the TBS protecting group as well as lactonization generated tricyclic lactone 9 (dr > 15 : 1) in 87% yield. It was noted that Et 2 AlCl removed the PMB protecting group at −78°C as evidenced by TLC analysis and then the IMDA reaction occurred at room temperature. Therefore, we believe that the excellent selectivity and the high yield of this transformation stem from the pre-formation of an aluminum-coordinated macrocycle intermediate (or transition state), which then undergoes an effective and endo-selective transannular Diels-Alder reaction. This result was remarkable regarding the yield, endo/exo selectivity and diastereoselectivity as compared to the previous IMDA reaction of tetraene substrates. [22][23][24]27 Regioselective dehydration of 9 was achieved by mesylation (MsCl/Et 3 N) and elimination with LiBr/Li 2 CO 3 /DMF 43,44 to afford trans-decalin 10 (92% yield), whose structure was conrmed by single crystal X-ray diffraction. We accomplished the rst stage of the efficient transdecalin synthesis and turned our attention to the second stage of the macrolactone synthesis. We rst explored BF 3 -Et 2 Omediated macrolactonization. 29 The lactone ring of decalin 10 was hydrolyzed with KOH and the resulting unstable hydroxyl carboxylic acid was treated with TMSCHN 2 to afford the corresponding ester 10 ′ , which was noticeably unstable under either mild acidic or basic conditions and should be oxidized immediately with DMP to provide aldehyde 11. While attempting to elaborate aldehyde 11 into (E,Z)-diene 14a by Stork-Zhao olenation and Stille coupling, 29 we encountered an unexpected difficulty in the formation of (Z)-vinyl iodide. Stork-Zhao olenation 45 of aldehyde 11 under various conditions led to a E/Z mixture of vinyl iodide (E/Z 1 : 1). Alternatively, we attempted Zselective Still-Gennari olenation 46  and partial hydrogenation with the Lindlar catalyst. Aldehyde 11 was converted into alkyne 12a with the Bestmann reagent, and then Sonogashira coupling with vinyl iodide 13a followed by Lindlar hydrogenation provided the desired (E,Z)-diene 14a in 64% yield over two steps. The installation of 1,3,5-tricarbonyls (14a / 16a) was challenging: Claisen condensation of ester 14a with 1,3-dicarbonyl 15a was not successful, while a four-step sequence (DIBAL-H reduction, DMP oxidation, Aldol reaction, and DMP oxidation) delivered 16a with 27% yield. Nevertheless, with tens of milligrams of 16a in hand, we explored the BF 3 ·Et 2 O-mediated macrolactonization, but we were not able to identify any desired product from many attempted reactions under various conditions (temperature and equivalent of BF 3 ·Et 2 O). TLC and NMR analysis clearly suggested that 16a decomposed in the presence of BF 3 ·Et 2 O. Then, we investigated the second macrocyclization strategy: RCM. Because Z-selective Stork-Zhao olenation failed to produce Z-vinyl iodide for subsequent Stille coupling, we decided to take a risk: Z-alkene formation by RCM as the last step, which contrasts with the previous RCM for Ealkene formation. 27 To verify this macrocyclization method, we prepared two substrates 16b and 16c, corresponding to anthracimycin and anthracimycin B, respectively. Wittig olenation of 11 followed by ester reduction with DIBAL-H and DMP oxidation afforded aldehyde 13b with 46% yield over 3 steps. The subsequent aldol reaction of 13b with dienolate of 1,3-dicarbonyl 14b/14c followed by DMP oxidation provided the key macrocyclization substrates 16b and 16c in 42% and 44% yield over two steps, respectively. The planned RCM of 16b and 16c proceeded smoothly with the Hoveyda-Grubbs II catalyst to furnish anthracimycin and anthracimycin B (exclusive Z-double bond), respectively, with >60% yield based on the crude NMR analysis (42-43% isolated yield). The NMR data of our synthetic samples were consistent with those reported for natural products (see ESI Tables S1-S4 †). Additionally, single crystals of anthracimycin were obtained for X-ray diffraction analysis, which further conrmed the structure of our synthetic anthracimycin.
Since our total synthesis required 14 steps, we set out to further optimize our synthetic route (Scheme 3). First, we employed N-phenyl tetrazole sulfone 4b, 47 instead of 4a (Scheme 2), for Julia-Kocienski olenation with aldehyde 3 so that the late-stage conversion of lactone to vinyl ester (9 / 12b, Scheme 2) was unnecessary (and thus saving us 4 steps). Reductive removal of the Evans chiral auxiliary and DMP oxidation of the resulting alcohol afforded aldehyde 5b (62% yield) with 6 : 1 E/Z selectivity for the newly-formed alkene. Next, we explored the possibility of one-pot sequential Mukaiyama vinylogous aldol (MVA)/intramolecular Diels-Alder (IMDA) reaction since both involved a Lewis acid as a promoter. Aer examining several acid promoters including TiCl 4 , BF 3 -Et 2 O, CBS-HOTf, proline, Rh(I) and B(C 6 F 5 ) 3 , we found that the sequential MVA and IMDA reactions could be achieved in one pot by treating the reaction mixture rst with B(C 6 F 5 ) 3 and then with Et 2 AlCl to provide a mixture of trans-decalins 8c and 8d (63% combined yield on a 1.1 g scale) with a 3 : 1 diastereomeric ratio in favor of the desired 8c. Although the diastereoselectivity of the IMDA reaction was moderate as compared to substrate 7 probably due to the lack of a macrocyclic intermediate (or a transition state) for the highly diastereoselective transannular Diels-Alder reaction, the overall efficiency was much higher. Dehydration with the Burgess reagent allowed DIBAL-H reduction of the ester to be carried out in one pot. DMP oxidation of the resulting alcohol provided aldehyde 13b (66% yield over two steps), which was elaborated to anthracimycin and anthracimycin B following the same protocol as in Scheme 2. The optimized route for 13b allowed us to accomplish the total synthesis of anthracimycin and anthracimycin B in 10 steps with 3.3-3.6% isolated overall yield from commercially available Evans chiral auxiliary 1 without using protecting groups and with only 7 isolated intermediates. The concise and efficient route enabled us to prepare 25 mg of anthracimycin and 14 mg of anthracimycin B, which allowed us to evaluate their antibacterial activity.
It is well recognized that the bioactivity of newly isolated natural products might be different from that of the corresponding synthetic samples (possibly due to contamination of natural  Table 1, synthetic anthracimycin and anthracimycin B were not effective against Gram-negative strains but they were potent against the Gram-positive bacteria (MIC: 0.03-0.8 mg mL −1 ), which is consistent with MIC values reported for natural anthracimycins. 15,17,18 A cytotoxicity study using HaCaT (cell line from human skin) suggested that both synthetic anthracimycins possessed low cytotoxicity (IC 50 > 14 mg mL −1 ), which holds great promise for further drug development. More interestingly, we found that both anthracimycins signicantly inhibited the MRSA biolm formation (MRSA ATCC29213) with MBIC values (MBIC 0.02 mg mL −1 and 0.6 mg mL −1 , respectively) even lower than the respective MIC values. As compared to vancomycin, a frontline antibiotic for treatment of MRSA infections, anthracimycin was 60 times more potent. This result is signicant because MRSA biolm formation 48-51 is primarily responsible for the reduced efficacy or inefficacy of many clinically used antibiotics for MRSA infections, and anthracimycin demonstrated potential as a new effective anti-MRSA antibiotic.

Conclusion
In summary, we have accomplished a 10-step asymmetric total synthesis of anthracimycin and anthracimycin B (the rst total synthesis) with 3.3-3.6% overall isolated yields. The key strategy is the use of alcohol as the latent alkene to enable convergent fragment coupling (vinylogous Mukaiyama aldol) and to enhance the endo/exo selectivity and yield of the intramolecular Diels-Alder reaction. Additionally, the ring-closing metathesis using the Hoveyda-Grubbs II catalyst allowed the efficient construction of a 14-membered macrolide with exclusive Zalkene selectivity. Our short and efficient synthetic route enabled us to prepare 25 mg of anthracimycin and 14 mg of anthracimycin B and allowed us to evaluate their antibacterial activities using our synthetic sample, which were consistent with those reported for the natural anthracimycins. Finally, we found that our synthetic anthracimycin signicantly inhibited the MRSA biolm formation with great promise to be a new effective anti-MRSA antibiotic.

Data availability
Experimental procedures and characterization data are available within this article and its ESI. † Data are also available from the corresponding author on request.

Author contributions
P. T., X. Z. and Y. T. performed the synthetic experiments. W. Y. performed the biological experiments. P. Q. and R. T. conceptualized and directed the project, and draed the manuscript with the assistance from all co-authors.

Conflicts of interest
The authors declare no competing interests.