Synthesis of bacterial 2-alkyl-4(1 H )-quinolone derivatives

The compound class of 2-alkyl-4(1 H )-quinolones represents a unique group of bacterial secondary metabolites that have been the subject of extensive research since their first discovery over 70 years ago. New insights into their structural diversity and their role in the complex interactions in bacterial ecology and human pathogenicity are still being discovered. In parallel with the ongoing discovery of new 2-alkyl-4(1 H )-quinolones and derivatives produced by microbes, synthetic methods were developed to facilitate access to these structurally diverse bioactive compounds. Here we present a detailed overview of the historical development and recent advances in their chemical synthesis


Introduction
Secondary metabolites have many important roles for microbial intra-and interspecies interactions.They serve for example as antibiotics against competing species, as virulence factors modulating host responses, or as signals to sense other cells of their own kin and trigger specific reactions in dependence of population density.Natural product isolation and structure elucidation have continuously increased the chemical space of microbial metabolites and shed light on the amazing biochemical capabilities of microbes.However, investigating their biological activity is often limited by the availability of sufficient quantities of pure compound.For this reason, bioassays typically focus only on a small set of antimicrobial and anticancer activities and a full functional characterization of many if not most microbial secondary remains elusive.A robust and scalable strategy for the synthesis of the corresponding compounds provides thus an important prerequisite to gain a more comprehensive understanding of the range of biological activities of a natural product.Some Pseudomonas and Burkholderia species feature biosynthetic gene clusters to produce derivatives of 2-alkyl-4(1H)-quinolones.These quinolones are produced in particular by Pseudomonas aeruginosa and members of the Burkholderia cepacia complex which are important pathogens for cystic fibrosis patients.Although the number of producing species is small, the diversity of quinolones produced by them is remarkable: P. aeruginosa produces more than 50 different quinolone derivatives. 1 This is achieved by a diversity-oriented biosynthetic machinery that allows the synthesis of different classes of quinolones and operates on a certain level of promiscuity to generate congeners with different chain lengths and saturation.
][4][5] These compounds have many interesting biological activities which are in part reviewed elsewhere. 6ome of them function as quorum sensing signals that allow their producers to coordinate virulence and other behaviours in dependence of population density.Others are mainly considered to act as anti-bacterial weapons deployed against competing species.Furthermore, some of these quinolones even mediate interkingdom interactions and impair the human immune response.
Investigating the diversity of the biological activities and functional specialization of different congeners requires robust strategies for the synthesis of these 2-alkyl-4(1H)-quinolone derivatives.Finally, these strategies will potentially allow to synthetically exploit these molecules for human use.Here we will review the approaches and developments described so far in the synthesis of bacterial 2-alkyl-4(1H)quinolones to the best of our knowledge.

Natural Diversity of 2-Alkyl-4(1H)-quinolones
The ability of Pseudomonas aeruginosa to produce quinolones had been already discovered in the 1950s.][9][10] Since then, a great diversity of bacterially produced quinolones has been characterized serving distinct and highly specialized biological purposes.In Pseudomonas aeruginosa these metabolites are produced by a series of enzymes encoded by the pqs gene cluster starting from anthranilic acid.The divergent biosynthetic pathway generates three classes of 4(1H)-quinolones: congeners of the Pseudomonas quinolone signal (PQS, 1), 2-alkyl-4(1H)-quinolone (AQs or pseudanes, 2), and 2-alkyl-4(1H)-quinolone N-oxides (AQNOs, 3).Since the enzyme complex PqsBC, which is responsible for quinolone biosynthesis, exhibits substrate promiscuity for diverse CoA-activated fatty acids, all classes are produced as mixtures of congeners with saturated and unsaturated C5-C11 alkyl chains. 113][14] PQS and its biosynthetic precursor HHQ are quorum sensing signals that are produced in dependence of population density and detected by the signal receptor PqsR (MvfR).][19][20] In contrast, AQNOs like HQNO do not participate in quorum sensing and have roles as weapons against competing species. 21In many Burkholderia species, a methyltransferase (HmqG) encoded in the biosynthetic gene cluster additionally results in the production of 3-methyl-AQs (MAQs, 4) or 2-methyl-AQNOs (MAQNOs, 5) (Figure 1). 22Some of these quinolones also feature double bonds with a different pattern such as AQs and AQNOs that mainly exhibit Δ 1 unsaturated alkyl chains and MAQs and MAQNOs that predominantly feature Δ 2 unsaturation.
Importantly, congeners of AQs, MAQs, AQNOs, and MAQNOs show functional differentiation with major differences in their biological activities.] Another class are the 2-geranylated 4(1H)-quinolones of Pseudocardia and Nocardia species, which include highly potent antibiotics against the intestinal pathogen Helicobacter pylori such as intervenolin (6, Figure 1). 2- 3Myxobacteria like Stigmatella and actinobacteria like Rhodococcus and Streptomyces produce aurachins, a structurally diverse class of compounds that among others also comprises 4(1H)-quinolones 10 (C-type aurachins) and their N-oxides.These aurachins are farnesylated in 3-position and feature a methyl-group in 2position and exhibit antibiotic activities mainly against gram-positive bacteria (Figure 1). 5 All bacterial 2-alkyl-4(1H)-quinolone derivatives also can be represented in their tautomeric form as corresponding 4-hydroxyquinolines (Figure 2).Under conditions of their synthesis or purification, we only observed the 2-alkyl-4(1H)-quinolone forms.However, the tautomeric equilibrium in aqueous media under microbial growth conditions has not been investigated so far.Several naming conventions of bacterial quinolones are therefore related to the tautomeric 4-hydroxyquinoline forms.For example, HHQ is the initial abbreviation for 2-heptyl-4-hydroxyquinoline.Also, the class of quinolone N-oxides is historically termed as such although the compounds are typically found in their 2-heptyl-1-hydroxy-4(1H)-quinolone form.3-Methylated 2-alkyl-4(1H)-quinolones (MAQs) are thus frequently termed HMAQs for 4-hydroxy-3-methyl-2alkenylquinolines and HMAQNOs for their corresponding N-oxides.
The corresponding β-keto esters can be obtained by the reaction of acid chlorides with Meldrum's acid in pyridine and subsequent alcoholysis under reflux conditions.Acid-catalyzed condensation of β-keto esters 11 with aniline affords the enamine tautomer of the Schiff base 12 that upon heating undergoes Conrad-Limpach cyclization.Finally, the 2-alkyl-4(1H)-quinolones 2 can be obtained in good yield and excellent purity by precipitation with non-polar solvents such as ether or n-hexane.The short reaction sequence and easy accessibility of the starting materials, β-keto esters and aniline, have certainly contributed to the success of this method.
Another strategy for the synthesis of 2-alkyl-4(1H)-quinolones 2 was published by Beifuss and Ledderhose. 31Here, they used N-Cbz protected quinolones 13 which were locked as 4-silyloxyquinolinium triflates 14 to direct the regioselective addition of alkyl-Grignard reagents on the 2-position.Subsequent deprotection of the Cbz-group with Pd/C and H2 yielded the corresponding 2-alkyl-4(1H)-quinolones 2 by an unexpected Saegusa-Ito-type oxidation reaction (Scheme 2).Scheme 3. Gold-catalyzed synthesis of HHQ via an α-iminogold carbene intermediate. 32ngh et al. developed a one-pot reaction for the synthesis of 2-substituted 4(1H)-quinolones.This reaction started from ortho-bromoaryl ynones 18, following tandem Michael addition of ammonia and Cu(I)mediated aryl amidation reaction.The ammonia was in situ generated from ammonium carbonate which also served as a base.This reaction allowed to synthesize various pseudanes (AQs) but also plant produced quinolones like waltherione F, graveoline and graveolinine (Scheme 4). 33heme 4. One-pot synthesis of HHQ and AQs via Michael addition and Cu(I)-mediated aryl amidation.
Recent work of the Clark group led to the discovery of several new quinolones with unusual side chains in 2-position which could be isolated from the Chinese P. aeruginosa strain BD06-03. 27These included unprecedented 4(1H)-quinolones with unsaturated branched side chain, methylthiovinyl 2h, and benzyl substitutions 2k.Ultimately, total synthesis reported by the same group gave access to some of these compounds for biological testing and revealed interesting activities against the growth of Staphylococcus aureus, Bacillus subtilis and even another strain of P. aeruginosa. 27,35 wo different synthetic routes were applied to the synthesis of the arylated and unsaturated quinolones and methylthiovinyl-substituted quinolones (Scheme 6).The methylthiovinyl side chain 2h was synthesized starting from a ketoaryl propiolamide 25 derivative before base-promoted Camps cyclization.Michael addition of methanthiolate on the alkynyl group of the propiolamide 26 yielded the methylthiovinyl substituted ketoaryl amide 27.Finally, the 4(1H)-quinolone 2k was obtained by base-promoted cyclization.In contrast, the benzyl and unsaturated side chain substituted 4(1H)-quinolones were obtained via coupling of the quinolone core 31, established by the Conrad-Limpach reaction, with the corresponding boronic esters via the Suzuki-Miyaura cross-coupling reaction. 35

Synthesis of the Pseudomonas Quinolone Signal (PQS)
. PQS 1 was discovered as a cell-to-cell signalling molecule that regulates virulence factor production and designated the term Pseudomonas quinolone signal (PQS).The first synthesis of PQS was described in 1999 along with its discovery by Pesci et al. 36 The reaction starts from HHQ 2a with the formylation of its 3-position by a Duff-reaction.Here, hexamine (urotropine) is applied as the formyl carbon source.Subsequent oxidation of the 3-formyl-2-heptylquinolone 32 via Dakin reaction with hydrogen peroxide afforded PQS 1 in 74% yield (Scheme 7).This method has been frequently used for synthesis PQS since then. 29A similar strategy was already reported in 1962 by Morgan et al.where Dakin oxidation of 3-formyl-4-hydroxyquinolines was applied in the synthesis of 2-phenyl and 2-methyl-3hydroxy-4(1H)-quinolones. 37heme 7. The first synthesis of PQS reported. 369] Formylation was only successful when HHQ 2a was previously converted into its 4hydroxyquinoline tautomer. 39 strategy to directly convert 4(1H)-quinolones to 3-hydroxy-4(1H)-quinolones was reported by Behrman et al. using an Elbs peroxodisulfate oxidation and acid-catalysed hydrolysis of the resulting sulfates. 40owever, the method has not yet been applied for the synthesis of natural 2-alkyl-3-hydroxy-4(1H)quinolones.
A facile and more direct strategy towards 3-hydroxylated quinolones 1 was described in 1999 by Hradil et al. 41 To this end, anthranilic acid ( 33) is esterified with α-chloro-or α-bromoketones giving the respective 2oxoalkyl 2´-aminobenzoates 34 in good yields.Cyclization is achieved by heating the esters with polyphosphoric acid at 120°C or under reflux with N-methylpyrrolidone (NMP) yielding the corresponding 2alkyl-3-hydroxy-4-quinolones 1 (scheme 8). 41This strategy was originally developed for non-natural 2-methyl and 2-phenyl 3-hydroxy-4(1H)-quinolones, but has since been adapted as a major route for the synthesis of PQS. 39,42 eme 8. Synthesis of 2-alkyl-3-hydroxy-4-quinolone according to Hradil et al. 41 Although the exact mechanism of cyclization in this reaction is not fully understood, a similar reaction to 2-phenyl-3-amino-4(1H)-quinolones gave a seven-membered diazepinone which rearranged to 2-phenyl-3amino-4-quinolone upon heating in polyphosphoric acid. 43An analogous rearrangement can be proposed for the synthesis of 3-hydroxy-4(1H)-quinolones. 38 similar strategy was developed by the Spring lab in order to achieve a facile synthetic access to a wide range of PQS 1 derivatives using a one-pot microwave-assisted synthesis of α-chloroketones 36 with anthranilic acid derivatives (Scheme 9). 38,44 hese conditions were further optimized for a continuous flow reaction that allowed the gram-scale synthesis of PQS and its derivatives. 38The resulting PQS analogues allowed to conduct a comprehensive investigation of structure-activity relationships for stimulating the quorum sensing receptor MvfR (PqsR). 45heme 9. Microwave-assisted and continuous flow synthesis of PQS analogues.

Synthesis of MAQs and MAQNOs of Burkholderia.
Species of the genus Burkholderia produce 2-alkyl-3methyl-4(1H)-quinolones (MAQs 4) and 2-alkyl-3-methyl-4(1H)-quinolone N-oxides (MAQNOs 5).4(1H)-Quinolones of the PQS-type have not been reported and the corresponding gene encoding for homologs of the monooxygenase PqsH is missing in Burkholderia.Recent work has shown that in addition to MAQs 4 and MAQNOs 5, Burkholderia thailandensis also produces lower amounts of the corresponding non-methylated AQs 2 and AQNOs 3. 24 While MAQs 4 are non-classical quorum sensing signals, 51 the corresponding MAQNOs 5 are potent antimicrobial compounds, the activity of which strictly depends on the exact pattern of unsaturation, methylation and position of the double bond of the congener. 24-Heptyl-3-methyl-4(1H)-quinolone (4a) was synthesized by Reen et al. by generating a methylated βketoester 11 with MeI and K2CO3 as base.Reaction with aniline to form the corresponding enamine followed by Conrad-Limpach cyclization of in diphenyl gave the final product (Scheme 14).29 Scheme 14. Synthesis of 2-heptyl-3-methyl-4(1H)-quinolone using a Conrad-Limpach cyclization.

Scheme 15. MAQ and MAQNO synthesis by Camps cyclization.
As one of the main quinolone N-oxides of Burkholderia thailandensis, trans-Δ 2 -MNQNO 5c was identified, which was synthesized by our laboratory along with trans-Δ 2 -NQNO 3e via the corresponding NQ and MNQ 4d derivative.The synthesis was achieved by coupling of an octenyl pinacol boronic ester with 2-(chloromethyl)quinolin-4(1H)-one 31 using a microwave assisted Suzuki-Miyaura reaction (Scheme 16). 24The same synthetic strategy was published in parallel in a collaboration of the Déziel and Gauthier laboratories for generating the three chain length congeners of trans-Δ 2 -MAQs and trans-Δ 2 -MAQNOs with heptenyl, octenyl, and nonenyl chains which demonstrated that trans-Δ 2 -MNQNO is the most active congener in antibiotic assays against various gram-negative and gram-positive bacteria. 52heme 16.Synthesis of trans-Δ 2 -unsaturated NQNO and MNQNO.
In addition, two 4(1H)-quinolones metabolites with hydroxyl groups in the side chain (8 and 9) had been reported from Pseudocardia sp.CL38489. 3 For the synthesis of these compounds, an alternative strategy to the Suzuki-Miyaura route had to be developed.Starting from geraniol (56), a MOM-protected propargylic alcohol 58 was generated which was subsequently used for Sonogashira coupling.Michael addition with methylamine and cyclization under Buchwald-Hartwig conditions gave the N-methyl 4(1H)-quinolone 61.Upon deprotection, a 1,3-allylic alcohol rearrangement gave rise to both hydroxylated natural products (Scheme 19). 54This strategy was also used to produce non-natural analogues of the 4(1H)-quinolones of Pseudocardia sp.CL38489, which exhibited activity in inhibition of pyocyanin production of P. aeruginosa. 55heme 19.Synthesis of the hydroxylated 4(1H)-quinolones of Pseudocardia sp.CL38489 (PPTS = pyridinium p-toluenesulfonate).

Synthesis of intervenolin of Nocardia
In 2013, Kawada et.al. discovered an N-iminodithiocarbonate-4(1H)-quinolone, named intervenolin (6), as a metabolite produced by the gram-positive bacterium Nocardia sp.ML96-86F2. 2Intervenolin exhibited antitumor activities and potently and selectively inhibited the growth of the gastrointestinal pathogen Helicobacter pylori. 2 Subsequently, total synthesis of intervenolin was developed combining Suzuki-Miyaura coupling with thiocyanate-isothiocyanate rearrangement as the key steps. 56The 4(1H)-quinolone core scaffold 62 was generated by Friedel-Crafts reaction via an anhydride produced by the reaction of the carboxylic acid 61 with Eaton's reagent (P4O10 dissolved in methanesulfonic acid), followed by TBS protection of the hydroxygroup.The quinolone was locked in its tautomeric quinolinediol form and activated as triflate 63 for subsequent Suzuki-Miyaura reaction (Scheme 20).The geranyl side chain at 2-position was introduced via the corresponding boronic ester 64.Thiocyanomethylation of the N-1 position followed by spontaneous rearrangement resulted in an isothiocyanate moiety 66.Finally, the isothiocyanate was captured by methyl thiolate and methylated by MeI to afford intervenolin (6). 56heme 20.Synthesis of intervenolin through Suzuki-Miyaura coupling. 56

Synthesis of 4(1H)-quinolones of the aurachin family
In 2013, Li et.al. reported the first synthesis of aurachin D (10a) through a three-step sequence starting from ethyl acetoacetate (67) which was farnesylated, condensed with aniline and cyclized to the 4(1H)-quinolone via the Conrad-Limpach reaction. 58

Conclusions
Bacterial 2-alkyl-4(1H)-quinolones are secondary metabolites that play an important role as quorum sensing signals and in the interaction between microbial species.Since their discovery and first isolation from Pseudomonas species, these quinolones have attracted great interest in their synthesis due to their diverse bioactivity, including antibacterial, antifungal, anti-malarial and anti-inflammatory activity.In this review, we have described the standard strategies as well as the most recent developments in the synthesis of microbial 4(1H)-quinolones, which have been divided into different subcategories according to their structural diversity.These strategies include many methods of synthesis ranging from traditional cyclization (Conrad-Limpach and Camps) to metal-catalyzed cyclization (Cu-and Au-catalyzed), and C-C cross coupling reactions (Suzuki-Miyaura and Sonogashira).Efficient synthetic methods have given access to this important group of microbial metabolites and enable more comprehensive studies of their biological roles and activities in quorum sensing, as virulence factors, and as antimicrobials.These methods will also inspire the synthesis of novel natural product-derived compounds with improved activity or selectivity for a relevant target species.

59 Scheme 21 .
Scheme 21.Synthesis of aurachins D and C via Conrad-Limpach and a reductive cyclization strategy.

Thomas
Böttcher studied chemistry and biochemistry at the Ludwig Maximilian University (LMU) of Munich.In 2009, he completed his Ph.D. at the LMU in the group of Prof. Stephan Sieber supported by a fellowship of the German Academic Scholarship Foundation.After a short postdoctoral stint at the Technical University of Munich, he co-founded the startup company AVIRU GmbH for preclinical drug-development.In 2011, he moved to Boston for postdoctoral research in the laboratory of Prof. Jon Clardy at Harvard Medical School on a Leopoldina Research Fellowship.In 2014, he started his independent research career at the University of Konstanz where he led an Emmy Noether research group and was a research fellow of the Zukunftskolleg.Since 2020, he is a full professor of Microbial Biochemistry, bridging the Institute of Biological Chemistry and the Department of Microbiology and Ecosystem Science at the University of Vienna.He received the Manfred-Fuchs-Prize 2019 of the Heidelberg Academy of Sciences and Humanities and an ERC Consolidator Grant in 2020.The research interests of his group include microbial secondary metabolites, customized antibiotics, as well as chemical modulators of bacterial behavior and synthetic inhibitors of virulence.This paper is an open access article distributed under the terms of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)