Iso-maleimycin, a Constitutional Isomer of Maleimycin, fromStreptomycessp. QL37

: Iso -maleimycin, a previously unknown constitutional isomer of the antibiotic maleimycin, has been detected in an extract of Streptomyces sp. QL37. Chemical synthesis of both maleimycin (20 % yield over seven steps) and iso -maleimycin, (15 % yield over six steps) allowed access to reference materials for identification. Gas Chromatography coupled Mass Spec-Introduction MIC Tests: The minimum inhibitory concentration (MIC) test was determined by the broth microdilution method using the British Standard BS EN ISO 20776–1:2006. [17,18] A stock solution of iso-maleimycin 5 was made by dissolving it in Mueller-Hinton (MH) broth to a concentration of 4 mg/mL. The bacterial indicator strains ( Escherichia coli ASD 19 and Bacillus subtilis ) were grown for approxi-mately two hours from an overnight culture until an OD 600 of 0.3 in MH broth and diluted until a concentration of bacteria of 1 × 10 6 CFU mL –1 in fresh broth. 50 μ L of MH broth were added to all the wells of the 96-well polypropylene microtiter plates. Then, 50 μ L of the sample stock was added to the first row to the concentration of 2000 μ g/mL, which was serially twofold diluted. Subsequently, 50 μ L of the indicator strains were added to the wells resulting in a range of antibiotic concentration from 1000 μ /mL to 7 μ g/mL. Three replicates were performed for each indicator bacterial strain. Growth control wells containing 100 μ L of 5 × 10 5 CFU mL –1 were included without test compounds. After overnight incubation at 37 °C, inhibition was defined as no visible growth compared to the growth observed in the control wells. For iso -maleimycin results were determined as 250 μ g/mL for E. coli ASD19 and 250 μ g/mL for B. subtilis . As comparison, for maleimycin the results were determined as 31 μ g/mL for E. Coli ASD19 and 125 μ g/mL for B. Subtilis . The authors thank T. Tiemersma-Wegman R. J. L. Sneep Groningen) the Organisatie voor Wetenschappelijk Onderzoek (NWO) financial


Introduction
Bacteria of the actinobacterial genus Streptomyces are known to produce a wide range of bioactive natural products, including antibiotics, anticancer compounds, and immunosuppressants. [1] These natural products include a range of maleimide-containing metabolites, of which showdomycin [2] (1) and maleimycin [3] (2) (Figure 1) are antibiotics produced by Streptomyces showdoensis. Maleimycin 2 was discovered independently after the discovery of 1, as it was detected by further analyzing UV absorption bands of culture extracts. [3] After isolation, the structure of 2 was elucidated mainly by 1 H-NMR, 13 C-NMR and mass spectrometry. [3] Although 1 and 2 originate from the same organism, feeding experiments with 3 H, 13 C and 14 C-labeled acetate and glutamate revealed that the formation of the maleimide ring in these compounds occurs via two different pathways. [3,4a] More recently, the showdomycin biosynthetic gene cluster (BGC) of S. showdoensis ATCC 15227 has been unraveled, by which a more detailed biosynthetic pathway of showdomycin has been proposed. [4b] A detailed biosynthetic pathway of 2 however remains unknown. Naturally occurring derivatives of maleimycin 2 have been isolated from S. nitrosporeus. [5] The diastereomeric nitrosporeusine A (3a) and nitrosporeusine B (3b) consist of a maleimycin moiety linked to a p-hydroxy-trometry (GC-MS) analysis demonstrated that of the two isomers, only iso-maleimycin was present in the extract. This finding supports our hypothesis that iso-maleimycin is a biosynthetic intermediate of lugdunomycin. Iso-maleimycin displays low antibiotic activity, with a Minimum Inhibitory Concentration (MIC) value on both E. coli and B. subtilis of 250 μg/mL. Recently, we reported the isolation and characterization of several previously undescribed angucycline derivatives in the extracts of Streptomyces sp. QL37, [7a] an antibiotic-producing actinomycete originating from mountain soil. [7b] Noticeable was the succinimide-containing compound lugdunomycin 4, an antibiotic with a rare chemical structure derived from the angucycline polyketide backbone. Angucyclines and their derivatives are the most diverse known family of polyketides, many of which have antibacterial and/or anticancer activity. [8a,b] Therefore, these compounds are of great interest for the pharmaceutical industry and for medical applications. During our investigations, a trace compound with the nominal mass of 2 was detected by LC-MS. As inspection of 4 suggested constitutional isomer 5, rather than 2, to be involved in its biosynthesis, [7a] we decided to determine the structure of this compound. Prelimi-nary supporting evidence for the presence of 2 or 5 was obtained by additional MS/MS fragmentation experiments and we dubbed the name of 5 to be iso-maleimycin. [7a] Structurally, the maleimycins 2 and 5 are composed of an uncommon bicyclic 5,6-dihydrocyclopenta[c]pyrrole-1,3(2H,4H)dione backbone. Maleimycin 2 carries a hydroxy function at the allylic C4 position and is chiral. A total synthesis of racemic 2 by Singh and Weinreb [6a] was reported in 1976, however assignment of the absolute configuration was performed later in synthetic studies by Philkana et al. [6c] in 2015. The configuration turned out to be R. In iso-maleimycin 5 the hydroxy group is positioned at C5, so as a result 5 is achiral.
As the compound of interest was detected only in small amounts, no attempt was made to isolate it. Instead we embarked on the chemical synthesis of both 2 and 5 to be able to compare and rigorously identify the structure of the natural material.

Synthesis of (rac)-Maleimycin
In the report by Weinreb and Singh, [6a] 2 was synthesized from 6 by allylic bromination, followed by nucleophilic substitution with silver trifluoroacetate and subsequent hydrolysis of the resulting trifluoroacetate ester (Scheme 1). Alternatively, 2 can be made by direct allylic oxidation with SeO 2 of 6. [6c,6d] Compound 6 in turn has been prepared starting from cyclopent-1-ene-1,2dicarboxylic acid (7a). [9] Amidation of 7a, followed by cyclisation would then yield 6. In turn, 7a can be obtained by cyclization of dimethyl α,α′-dibromopimelate 10 with sodium hydride in DMF, followed by acidic hydrolysis of the resulting diester (dimethyl cyclopent-1-ene-1,2-dicarboxylate). [9] Compound 10 can be readily obtained by Hell-Vollhardt-Zelinski bromination of pimelic acid. [9] Unfortunately, when we attempted the sodium hydride mediated cyclisation procedure of 10 on a multigram scale, the main product isolated was the isomeric dimethyl cyclopent-2-ene-1,2-dicarboxylate. Therefore, we sought for alternative strategies to arrive at 7a/b. In the literature [10a,10b] it has been described that ethyl ester 7b can be obtained in high yield, in two steps, starting from 8. These steps include the conversion of 8 into its corresponding enol triflate, which is then transformed into 7b by means of a palladium catalyzed carboxylation reaction. [10a,10b] Indeed, commencing the synthesis of compound 2 (Scheme 2), enol triflate 11 was readily prepared from Dieckmann product 8 in 94 % yield, by treatment with sodium hydride and trapping the resulting enolate with Tf 2 O. [10a,10b] Enol triflate 11 was subjected to a palladium catalyzed carboxylation, as reported by Yoshimitsu et al. [10a] Treatment of 11 with sodium formate, acetic anhydride and catalytic (5 mol-%) Pd(OAc) 2 in the presence of LiCl provided carboxylic acid 7a in near quantitative yield. As anticipated, 7a could be converted into amide-ester 12 by exposure of the corresponding acid chloride to concentrated aqueous ammonia under Schotten-Baumann conditions. [11] This procedure gave amide 12 in 86 % yield.
Subsequently, cyclization conditions were investigated in order to arrive at the desired imide 6. Due to the presence of considerable ring strain in 6, cyclisation of the imide-precursor requires forcing conditions. Weinreb and Singh [6a] obtained 6 by a two step approach; first converting the acid-amide, derived from compound 7b, into the corresponding nitrile, and then cyclisation to 6 in refluxing trifluoroacetic anhydride. We aimed for the direct conversion of 12 into 6 instead. It was hypothesized that cyclization would occur after deprotonation of amide 12 with a strong base. Fortunately, when a solution of 12 in THF was added to NaH, it underwent immediate conversion into imide 6. It was observed by analytical TLC, however, that some polar side-products were formed, presumably caused by degradation or polymerisation of the starting material. Compound 6 was isolated in an appreciable 60 % yield. This result is comparable with the two-step procedure reported previ-ously. [6a-6d] With 6 in hand we completed the synthesis according to the procedure of Weinreb and Singh [6a] to obtain racemic maleimycin. Maleimycin 2 was prepared in 20 % overall yield in seven steps.

Synthesis of Iso-maleimycin 5
It was envisioned (Scheme 3) that the maleimide unit in 5 could be constructed using the same cyclisation strategy as in the synthesis of 2. This required the synthesis of 14a or 14b, in turn planned to be prepared from commercially available 15, by reduction of the ketone followed by introduction of the double bond.
The synthesis of iso-maleimycin (5) (Scheme 4) commenced with the reduction of commercially available rac-15 with NaBH 4 in methanol. The resulting crude 16 was directly treated with TBDPSCl and imidazole in DMF, [12] to provide 17 in 77 % yield based on 15. A key step was the preparation of compound 14b by the introduction of the double bond. According to literature, [13] 17 was treated with an excess of LDA to form the mono-enolate, which was treated with a stoichiometric amount of iodine in THF at -78°C to generate the corresponding iodinated intermediate. This intermediate then likely reacted with a second equivalent of LDA providing the corresponding elimination product 14b, which was isolated in 74 % yield.
The next phase was to access 18. Based on previous experiments, it was expected that mono-amide 18 could be prepared from mono-acid 14a, which in turn was envisioned to be obtained from hydrolysis of compound 14b. It was found that 14b could be hydrolyzed with potassium hydroxide in a water/THF/ MeOH mixture, yielding mono-acid 14a in 64 % yield. Subsequently 14a was converted into mono-amide 18, using the same Schotten-Baumann procedure used in the synthesis of 9. [11] With this approach, the mono-amide was obtained in 54 % yield, as tarry products were formed in significant amounts. To reduce loss of material throughout the synthesis, an alternative single-step procedure was sought for the conversion of diester 14b into mono-amide 18. Weinreb and co-workers [14] had reported that aluminium amide complexes of the type [AlCl(CH 3 )NR 2 ] (R = H or alkyl) can be used to convert esters into their corresponding amide. The reagent [AlCl(CH 3 )NH 2 ] was generated from ammonium chloride and Al(CH 3 ) 3 (as a solution in toluene), to which 14b was added subsequently. We were delighted to observe that in this way 18 was prepared in 53 % yield directly from 14b.
With a sufficient amount of 18 in hand, cyclisation to the corresponding imide 19 was studied. As expected, when 18 was treated with NaH in THF at 0°C, 19 was obtained in 65 % yield. We occasionally observed that upon addition of the substrate to the sodium hydride suspension, the reaction did not initiate spontaneously. We speculate this is caused by the poor solubility of sodium hydride in THF. The issue could be readily solved by pre-mixing 18 with 1.3 equiv. of 15-crown-5 before adding it as a solution in THF to the sodium hydride, affording the desired product without affecting the yield. It has been proposed that 15-crown-5 acts as a phase transfer reagent aiding solubilization of sodium hydride, thereby increasing its basicity. [15] Due to immediate initiation in presence of 15-crown-5, we anticipate that at larger scale potential runaway reactions can also be prevented. Finally, desilylation of 19 was performed using TBAF in THF, [12] which gave iso-maleimycin 5 in 76 % yield. Overall, over the shortest sequence in six steps, compound 5 was obtained in 15 % yield.

Identification of Iso-maleimycin 5 in Extracts of S. sp QL37, Using GC-MS
Streptomyces sp. QL37 was isolated from soil in the Qinling mountains (P. R. China) and deposited to the collection of the Centraal Bureau voor Schimmelcultures (CBS) in Utrecht, The Netherlands, under deposit number 138593. [7a,7b] The extract was prepared as indicated in the experimental section. GC-MS was selected for the analysis due to the low molecular mass and hence relatively volatile nature of 2 and 5. In addition, structural information can be obtained from the EI fragmentation spectra corresponding to the TIC chromatograms. The analysis was performed by injecting samples of compound 2 and compound 5 (0.23 mg/mL), and also a sample of the extract (1 mg/mL). It was found by comparing the chromatograms and their corresponding EI mass spectra that S. sp. QL37 produces iso-malei-   [16] .
Eur. J. Org. Chem. 2020, 5145-5152 www.eurjoc.org © 2020 The Authors published by Wiley-VCH GmbH 5148 mycin 5 but not maleimycin 2. In the TIC chromatogram (Figure 2) of the isolate, based on retention times two signals were identified which potentially corresponded to 2 or 5. Closer inspection and comparison with synthetic 5 indicated that this compound is present in the extract, which was confirmed by matching fragmentation spectra ( Figure 3).

Antibiotic Assays
Since compound 2 is a known antibiotic, [3,6] it was anticipated that compound 5 might exhibit similar properties due to its Full Paper doi.org/10.1002/ejoc.202000767

EurJOC
European Journal of Organic Chemistry closely related structure. 5 Was tested for antibiotic activity, and it was found that 5 displayed growth inhibition in a disk diffusion assay (1000 μg/mL) on Escherichia coli and Bacillus subtilis. Following these results, the Minimum Inhibitory Concentrations (MIC) [17,18] for 5 were determined. The MIC of 5 was found to be 250 μg/mL for E. coli ASD219 and 250 μg/mL for B. subtilis. MIC values for (rac)-2 were also determined for comparison, and were found to be 31 μg/mL on E. coli and 125 μg/mL for B. subtilis; indicating that 2 is a more potent antibiotic than 5.

Conclusion
In conclusion, iso-maleimycin 5, a maleimide-containing metabolite, and a constitutional isomer of maleimycin 2, has been identified in Streptomyces sp. QL37. 5 has been synthesized, affording the product in 15 % overall yield over six steps. Additionally, maleimycin 2 was also synthesized according to a modified synthetic sequence and the compound was obtained in 20 % overall yield over seven steps. Comparison of the synthetic compounds by GC-MS with an extract of S. sp. QL37 confirmed unambiguously that iso-maleimycin is produced by S. sp. QL37. However, the previously described maleimycin 2 was not detected in the extract. Detection of 5 serves as evidence for our hypothesis that it is a biosynthetic precursor of 4. Currently we are interested in the exact biosynthetic origin of 5, its relationship with maleimycin, and its role in the biosynthesis of lugdunomycin 4. Antibiotic assays of 5 showed that it displays weak antibiotic activity towards gram positive B. subtilis and gram negative E. coli as the MIC values were found to be 250 μg/mL. In these assays iso-maleimycin 5 is less potent than maleimycin 2.

Experimental Section Synthetic Chemistry Experiments
General: All moisture and oxygen sensitive reactions were executed under a N 2 atmosphere. All reaction solvents were purchased from commercial vendors and used without further purification unless specified otherwise. Reagents were purchased from chemical vendors and used without further treatment or purification, unless stated otherwise. NMR spectra were recorded on an Agilent 400 NMR spectrometer, detected 1 H-nuclei at 400 MHz, 13  Ethyl 2-Carbamoylcyclopent-1-ene-1-carboxylate (12): According to a modified literature procedure, [11] crude mono-ester 7a (

5,6-Dihydrocyclopenta[c]pyrrole-1,3(2H,4H)-dione (6):
A roundbottomed flask was equipped with a stir bar and NaH 60 % suspension in mineral oil (482 mg, 12.1 mmol) which was rinsed with pentane three times. A solution of 12 (1.70 g, 9.28 mmol) in 44 mL of THF was added dropwise via syringe, while cooling the reaction mixture on ice. After 15 min, when H 2 evolution had ceased, the reaction mixture was quenched by adding acetic acid (0.7 mL). The mixture was diluted with water and extracted with EtOAc (3 × 50 mL), the combined organic layers were dried with MgSO 4 and concentrated in vacuo. The product was purified by means of column chromatography (EtOAc/pentane, 25:85), to give the pure imide as a white crystalline solid (765 mg, 5.58 mmol, 60 % yield). Alternatively, an analytically pure sample was obtained by means of recrystallization from CHCl 3 /pentane (50:50) and cooling to -78°C, in comparable yield and purity.

Methyl 4-[(tert-Butyldiphenylsilyl)oxy]-2-carbamoylcyclopent -1-ene-1-carboxylate (18)
Method 1, direct conversion of diester 14b. According to a modified procedure, [14] ammonium chloride (331 mg, 6.19 mmol) was suspended in toluene (10 mL). While cooling the suspension in an ice bath, 2 M trimethylaluminium (solution in hexanes, 3.1 mL, 6.19 mmol) was added dropwise, after which evolution of methane was observed. The ice bath was removed and the reaction mixture was stirred at r.t. for 3 h. A solution of 14b (905 mg, 2.06 mmol) in toluene (10 mL) was added dropwise to the reaction mixture, subsequently the mixture was heated to 50°C and stirred for 18 h. The reaction mixture was cooled in an ice bath and quenched with 1 M HCl aq (15 mL) and stirred for 15 min. The resulting clear biphasic mixture was extracted with EtOAc (5 × 15 mL), the combined organic layers were dried with MgSO 4 and concentrated in vacuo. The crude product was purified by column chromatography, (pentane/ EtOAc, 60:40) affording 18 (467 mg, 1.10 mmol, 53 % yield) as a pale brown crystalline solid.
Method 2, from acid-ester 14a. Following a modified literature procedure, [11]  with a column flow of 0.9 mL/min, pressure 82.7 kPa. Ionization by means of EI, ionization energy 70 eV, mass range from m/z 50-225. The identification was done by comparing retention times and mass fragmentation patterns of the synthetic standards with those obtained from the extracts.

Biological Assays
Disc Diffusion Assay with Soft Agar Overlay: The antimicrobial properties of iso-maleimycin were determined by disc diffusion assay. Briefly, three colonies of the indicator strains, Bacillus subtilis and Escherichia coli ASD19, were picked from an agar plate for the inoculation of an overnight culture. Afterwards, 300 μL of the overnight culture were added in 10 mL of fresh LB broth and incubated at 37°C until a OD 600 of 0.3. Then, a LB plate was overlaid with LB soft agar (0.75 % w/v agar) containing 1.5 mL from one of the indicator strains pre-grown in liquid LB to OD 600 of 0.3. When solidified, antibiogram discs loaded with 10 μL of 1 mg/mL of iso-maleimycin were applied on it and incubated overnight at 37°C. Ampicillin 1 μg/mL was used as a control.

MIC Tests:
The minimum inhibitory concentration (MIC) test was determined by the broth microdilution method using the British Standard BS EN ISO 20776-1:2006. [17,18] A stock solution of isomaleimycin 5 was made by dissolving it in Mueller-Hinton (MH) broth to a concentration of 4 mg/mL. The bacterial indicator strains (Escherichia coli ASD 19 and Bacillus subtilis) were grown for approximately two hours from an overnight culture until an OD 600 of 0.3 in MH broth and diluted until a concentration of bacteria of 1 × 10 6 CFU mL -1 in fresh broth. 50 μL of MH broth were added to all the wells of the 96-well polypropylene microtiter plates. Then, 50 μL of the sample stock was added to the first row to the concentration of 2000 μg/mL, which was serially twofold diluted. Subsequently, 50 μL of the indicator strains were added to the wells resulting in a range of antibiotic concentration from 1000 μ/mL to 7 μg/mL. Three replicates were performed for each indicator bacterial strain. Growth control wells containing 100 μL of 5 × 10 5 CFU mL -1 were included without test compounds. After overnight incubation at 37°C, inhibition was defined as no visible growth compared to the growth observed in the control wells. For iso-maleimycin results were determined as 250 μg/mL for E. coli ASD19 and 250 μg/mL for B. subtilis. As comparison, for maleimycin the results were determined as 31 μg/mL for E. Coli ASD19 and 125 μg/mL for B. Subtilis.