The Epoxybenzooxocine Core of Integrastatins and Analogs: A Comprehensive Review of Structure, Synthetic Approaches, and Biological Applications

Abstract

Integrastatins and their biogenetic counterparts possess a rare heterotetracyclic framework containing an unprecedented [6/6/6/6]-tetracyclic skeleton and a central [3/3/1]-bicyclic ketal core with high pharmaceutical potential, particularly due to their potent anti-HIV-1 integrase activity. Despite their promising scaffolds, a systematic consolidation of their chemistry and biology has been entirely lacking, creating a critical gap between the complex architecture of these natural polyketides and the targeted design of drug-like inhibitors. Herein, we present the first comprehensive review of this field, systematically analyzing 42 peer-reviewed publications from 1999 to 2025 retrieved from the Scopus, Web of Science, PubMed, and PubChem databases. Structural analysis confirms that while these rigid, nonplanar V-shaped molecules comply with Lipinski’s Rule of Five as a baseline filter for drug-likeness, deeper ADMET profiling further highlights their high predicted human intestinal absorption (HIA) alongside a low risk of cardiotoxicity. Synthetically, we detail how the field has fundamentally evolved from an arduous 11-step sequence in 2003 to highly streamlined, atom-economical one-step protocols in 2008 and 2021, culminating in the 2025 total syntheses that successfully resolved long-standing structural misinterpretations of epicoccolide A and epicocconigrone A. Furthermore, a comparative assessment across eight critical synthetic parameters highlights the operational trade-offs between step efficiency, scalability, and functional group tolerance in current cascade cyclizations. Biologically, we evaluate the extensive in vitro and in silico profiling of over 30 synthetic derivatives, highlighting their antimicrobial (M. bovis, S. aureus, P. carotovorum, C. albicans), antitubercular, and antiviral (SARS-CoV-2 and HIV-1 integrase inhibition) potencies. Integrating pharmacophore mapping with in silico ADMET profiling demonstrates that despite favorable absorption and cardiac safety, predicted liabilities such as human hepatotoxicity, carcinogenicity, and skin sensitization highlight critical areas for targeted structural modification. Ultimately, by bridging synthetic methodology with in silico and experimental bioactivity evaluation, this review establishes epoxybenzooxocine-based heterocycles as viable early-stage lead platforms and outlines strategic directions to advance these scaffolds in targeted drug discovery.

1. Review Plan and Methodology

This review presents a comprehensive analysis of advances in the synthesis of integrastatin-type heterocycles, aiming to bridge the gap between the complex architecture of natural polyketides and the targeted design of potent, drug-like HIV-1 integrase inhibitors. The primary objective of this review was to fill a gap in the systematic literature on the design of integrastatin-like heterocycles.

This is the first comprehensive review devoted to natural integrastatins and their synthetic analogues, with an emphasis on synthesis, structure, and biological activity. It includes a detailed description of the discovery process of integrastatins and their analogues with an epoxybenzooxocine core, a systematic analysis of various synthesis approaches with an emphasis on possible biosynthetic pathways, historical multistep and recent one-step strategies. Particular attention is paid to the functionalization of epoxybenzoxocine derivatives, their diverse biological activity, and prospects for application in medicine. These include antibacterial, antifungal, and antitubercular activities, as well as cytotoxicity and antiviral activity against SARS-CoV-2 and HIV-1 integrase.

The reviewed literature covers publications from 1999 to 2026, reflecting the main trends and achievements in this promising field from the discovery of integrastatins to the present day. In preparing this review, we also made extensive use of our own publications in this field, supplemented by a comprehensive review of peer-reviewed sources from Scopus, Web of Science, PubMed, and other academic databases. The literature search was conducted using targeted keywords, including “integrastatin,” “epicoccolide,” “epicocconigrone,” “epoxybenzooxocine,” and “[6/6/6/6]-tetracyclic skeleton.” Despite the broad scope of this subject, there are currently no review articles. This review fills this gap.

The following criteria were prioritized in selecting sources for this review: original scientific papers devoted to the discovery of natural integrastatins; important steps in the total synthesis of the tetracyclic core; and recent publications (2020–2025) detailing new one-step synthesis methods and the functionalization of epoxybenzooxocine derivatives. This review is based on an analysis of 42 key scientific papers, supplemented by recent findings obtained through in silico computational tools and molecular docking studies against target viral proteins. The IUPAC nomenclature of the compounds was confirmed in the PubChem open chemistry database of the National Institutes of Health (NIH) (https://pubchem.ncbi.nlm.nih.gov/ (accessed on 29 March 2026). All chemical structures were constructed using ChemDraw Ultra 22.2.0 software. Physicochemical drug-likeness profiles and ADMET parameters were predicted in silico using the AdmetLab 3.0 online platform (https://admetlab3.scbdd.com/server/).

2. Introduction

The search for new antiretroviral and antiviral drugs remains a global health priority, particularly in the context of the ongoing HIV-1 epidemic and the emergence of SARS-CoV-2. − Among the various viral enzymes, HIV-1 integrase is a critical target because it has no cellular counterpart in humans, meaning that its inhibition is unlikely to disrupt normal cellular metabolism.

Integration is a critical step in the life cycle of retroviruses, including HIV, and is a three-step process involving assembly of proviral DNA by integrase, endonucleolytic processing of proviral DNA, and transfer of the proviral DNA strand into the host cell DNA. This is a unique process by which the virus replicates, all catalyzed by a single viral enzyme, HIV-1 integrase. This process, like the enzyme itself, is absent in the host organism, making it a safe target for the development of new anti-HIV therapies. The recent discovery of diketo acid-based integrase inhibitors, which demonstrated comparable antiviral activity to that of clinically effective protease inhibitors, has supported the development of integrase inhibitors as effective therapeutic agents. , Although diketo acids represent promising small-molecule chemistries, the unpredictable nature of the drug development process makes it important to explore alternative structures that could potentially inhibit integrase.

Extracts of natural compounds have historically been used in the search for potential drug candidates because they are inherently biocompatible, generally less toxic, and often possess unique structural frameworks that provide chemical diversity exceeding that of typical synthetic libraries. , Screening of such extracts for recombinant HIV-1 integrase led to the discovery of several new natural inhibitors, including equisetin, integric acid, and complestatin. Continued screening of fungal extracts led in 2002 to the discovery of two racemic compounds named integrastatins A and B (Figure ).

1.

Integrastatins R = CH₂OH (Integrastatin A); R = CHO (Integrastatin B).

These tetracyclic aromatic [6/6/6/6/]-heterocycles of polyketide origin were shown to inhibit the strand transfer reaction of recombinant HIV-1 integrase, with IC50 values of 1.1 and 2.5 μM, respectively. However, their practical application has been seriously limited by low abundance in endemic natural fungal sources and the high cost of isolation. Therefore, since their initial discovery in 2002, the development of efficient methods for synthesizing these structural frameworks and their structural analogues has become a major area of organic chemistry.

3. Discovery and Structural Architecture of Natural Integrastatins and Their Analogs with an Epoxybenzooxocine Core

In 2002, Singh and coworkers at Merck Laboratories isolated Integrastatin A (2a) and Integrastatin B (2b) for the first time from an unnamed fungal source (ATCC 74478) and an Ascochyta species (ATCC 74477). They were found to be natural heterocyclic compounds containing an epoxybenzooxocine core (Figure ).

2.

Structural formulas of Integrastatins A (2a) and B (2b), Epicoccolide A (3), Epicocconigrone A (4) and Epicoccolide B (5) .

It was also noted that both of these integrastatins contain an unprecedented [6/6/6/6]-tetracyclic skeleton and two chiral centers; in nature, they exist as a racemic mixture. In a preliminary screening using recombinant HIV-1 integrase, integrastatins were found to inhibit the strand transfer reaction (IC₅₀ = 1.1 μM for integrastatin A and 2.5 μM for integrastatin B). , The unique structural architecture of integrastatins, combined with the critical demand for new inhibitors of HIV replication, has made these compounds attractive targets for total synthesis and drug development.

The isolation of two new natural compounds, epicoccolide A (3) and epicocconigron A (4), by the research groups of Laatsch and Proksch in 2013 has rekindled interest in tetracyclic structures with an epoxybenzooxocine core as promising pharmacophores. These natural compounds have demonstrated a wide range of biological activities, including anticancer, antimicrobial, and antifungal effects. Epicoccolide B (5) was identified as the common biogenetic partner of compounds (3) and (4) (Figure ).

Structurally, Integrastatins A and B, Epicoccolide A (3), and Epicocconigrone A (4) are tetracyclic aromatic heterocycles with an unprecedented [6/6/6/6]-tetracyclic skeleton consisting of a central [3/3/1]-bicyclic ketal core connected to aromatic rings on both sides. A critical structural differentiation is observed in the oxidation state of the substituent at the C-12 position: Integrastatin A (2a) features a hydroxymethyl group (−CH₂OH); Integrastatin B (2b), Epicoccolide A (3), and Epicocconigrone A (4) possess a formyl group (−CHO). These compounds typically adopt a nonplanar V-shaped or inverted V-shaped conformation due to the rigid structure of the oxocine ring (Figure ).

3.

3D conformers of Integrastatins A (2a) and B (2b), Epicoccolide A (3), and Epicocconigrone A (4).

As shown in Figure , the relative stereochemistry and conformation of integrastatins A (2a) and B (2b) were determined to be (R,R) or (S,S). These models showed that the bridging oxygen connects the C-2 and C-10 atoms via diequatorial bonds, providing the lowest-energy conformation and thus leading to diaxial fusion of the pyranone ring to form an inverted V-shaped (R,R) or V-shaped (S,S) conformation. This results in only equatorial orientations of the C-18 and C-19 methyl groups and the formation of wing-like structures. Epicoccolide A (3) and epicococonigraone A (4) are characterized by the same V-shaped conformation, but in Epicocconigraone A (4), an intramolecular hydrogen bond is formed between OH-7 and the adjacent carbonyl (C-9).

Integrastatins A (2a) and B (2b), epicoccolide A (3), and epicocconigron A (4) are currently indexed in the PubChem database, providing access to both their structural data and physicochemical profiles. − Their key molecular descriptors, such as molecular formula and mass, lipophilicity (XLogP3-AA), number of hydrogen bond donors (HBD) and acceptors (HBA), number of rotatable bonds, topological polar surface area (TPSA), number of heavy atoms, and molecular complexity, can be used for general characterization of their physicochemical properties and bioactive potential (Table ). Compliance with Lipinski’s Rule of Five (Ro5) serves merely as a baseline filter for chemical space and does not guarantee optimal oral absorption or a favorable in vivo PK/PD profile.

1. Molecular Descriptors of Integrastatins A (2a) and B (2b), Epicoccolide A (3), and Epicocconigrone A (4) from the PubChem Database.

Descriptor	Integrastatin A (2a)	Integrastatin B (2b)	Epicoccolide A(3)	Epicocconigrone A (4)	Ro5 Limit
PubChem CID	10046792	464001	71732639	73891099
Molecular Formula	C20H20O9	C20H18O9	C18H14O9	C18H14O9
Molecular Weight, g/mol	404.4	402.4	374.3	374.3	≤500
XLogP3-AA	1.1	1.4	1.1	1.7	≤5
Hydrogen Bond Donor (HBD) Count	4	3	5	5	≤5
Hydrogen Bond Acceptor (HBA) Count	9	9	9	9	≤10
Rotatable Bond Count	3	3	1	1	≤10
Topological Polar Surface Area (TPSA), Å2	135	132	154	154	≤140
Heavy Atom Count	29	29	27	27
Complexity	658	685	626	626
Lipinski’s Rule of Five (Ro5) violations	0	0	0	0

^aRo5 - Lipinski’s Rule of Five.

^bA TPSA value of ≤140 Ų is consistent with Veber’s rules for favorable oral bioavailability.

The data in Table show that Integrastatins A (2a) and B (2b), Epicoccolide A (3), and Epicocconigron A (4) represent a class of highly oxygenated tetracyclic compounds with various structural modifications that influence their physicochemical properties and potential biological interactions. Integrastatins A and B are characterized by a higher degree of alkylation and methoxylation, which is reflected in their molecular formulas (C₂₀H₂₀O₉ and C₂₀H₁₈O₉, respectively) and molecular weights of 404.4 and 402.4 g/mol. In contrast, Epicoccolide A and Epicocconigron A are structural isomers (C₁₈H₁₄O₉, 374.3 g/mol) characterized by a higher degree of hydroxylation due to methoxy groups. It is noteworthy that the molecular weights of these compounds do not exceed the threshold value of 500 g/mol, which satisfies the key criterion of Lipinski’s rule of five regarding oral bioavailability

The pharmacokinetic potential of the molecules under study is determined by key characteristics such as Topological Polar Surface Area (TPSA) and lipophilicity (XLogP3-AA). Epicoccolide A and Epicocconigron A exhibit the highest TPSA values of 154 Ų, well above the threshold of 140 Ų often associated with low cell membrane permeability. This is consistent with their high HBD count (5), suggesting a strong potential for hydrogen bonding but possibly limited passive diffusion. Integrastatins A and B exhibit more moderate TPSA values of 135 and 132 Ų, respectively, placing them closer to the preferred range for drug-like candidates. Their XLogP3-AA values (1.1–1.4) indicate a relatively hydrophilic nature, which may facilitate solubility but may require specific transport mechanisms for intracellular targets.

Molecular complexity indices in PubChem quantify the information content of a molecular graph by taking into account connectivity, elemental diversity, and stereochemical features, using the standardized Bertz-Dalby algorithm. , The molecular complexity indices for compounds 2–4, ranging from 626 to 685, highlight the complex stereochemical architecture of these metabolites.

Overall, the comparative analysis of the molecular descriptors in Table of compounds 2–4 suggests several key conclusions regarding their pharmaceutical potential. First of all, all three natural products 2–4 strictly comply with Lipinski’s Rule of Five, without any violations. Their molecular weights remain well below 500 Da, and their lipophilicity (logP) values indicate a balanced hydrophilic–lipophilic nature, which is favorable for membrane permeability. Notably, although epicoccolide A (3) and epicocconigron A (4) reach the upper limit for hydrogen bond donors (HBD = 5) and slightly exceed the conventional TPSA threshold of 140, the overall profiles indicate that these tetracyclic structures have a high degree of drug-likeness and represent excellent candidates for further structural optimization. The presence of the formyl group in compounds 2, 3, and 4 provides a reactive “handle” for further semisynthetic modifications, allowing the creation of Schiff bases or other derivatives to fine-tune biological activity. The significant number of hydrogen bond acceptors (HBA = 9) in molecules 2–4 indicates a high capacity for anchoring in protein binding pockets, especially those involved in enzyme inhibition, such as HIV-1 integrase or telomerase. Integrastatin B (2b) with the lowest TPSA and optimal lipophilicity, appears to have the most balanced profile between structural complexity and physicochemical properties. At the same time, the high degree of hydroxylation of epicoccolide A (3) and epicocconigron A (4) makes them excellent candidates for free radical scavenging studies. Although the number of heavy atoms is not a formal criterion for Lipinski’s Rule of Five, the values for compounds 2–4 (27–29 atoms) fall within the optimal range for searching for potential drug compounds, confirming their potential for further lead optimization. However, compliance with Lipinski’s Rule of Five (Ro5) serves merely as a baseline filter for chemical space and does not guarantee optimal oral absorption or a favorable in vivo PK/PD profile.

In summary, natural compounds 2–4 represent a promising scaffold for the development of targeted therapeutics. However, since the main natural sources of these compounds are endemic fungi, their availability is significantly limited, hindering potential industrial scaling. This shortage has made the development of efficient synthesis methods extremely necessary, and several research groups around the world have focused on finding optimal routes. In the following sections of this review article, we will examine current progress and challenges in the chemical synthesis of these building blocks, with particular emphasis on the transition from complex multistep syntheses to increasingly simpler ones.

4. Synthetic Approaches to the Epoxybenzooxocine Core

A systematic search of Scopus, Web of Science, PubMed, and other academic databases revealed a limited number of publications devoted to the synthesis of the epoxybenzooxocine core of the integrastatin molecule. Notably, these studies were published in reputable chemical journals such as Organic Letters, Chemical Communications, Organicand Biomolecular Chemistry, and the Journal of Organic Chemistry, highlighting the high relevance and complexity of synthesizing this structural motif. −

4.1. Possible Biosynthetic Pathways for the Formation of the Epoxybenzooxocine Core

Given the structural complexity and the scarcity of natural sources, the development of efficient laboratory methods for the synthesis of integrastatins and their analogues is of paramount importance. A rational approach to designing these synthetic routes often draws inspiration from the proposed biosynthetic origins of these molecules. In the first paper on integrastatins, Singh proposed that their biogenesis occurs via the traditional polyketide pathway (Scheme ).

1. Proposed Biogenesis of Integrastatins (2a–b) .

It was proposed that the main carbon skeleton is formed by the condensation of nine acetate units to form a poly-β-ketone chain. The formation of a complex tetracyclic system is further proposed to occur through subsequent enzymatic transformations, including regioselective aromatization, decarboxylation, and specific methylation and oxidation steps.

The proposed biosynthetic pathway for epicoccolide A (3) was presented by Talontsi as a more direct alternative to the traditional nonaketide route (Scheme ).

2. Proposed Biosynthetic Pathways to Epicoccolide A (3) .

While integrastatins were previously thought to derive from nine acetate units through a sequence of decarboxylation, C-methylation, and oxidation, the formation of Epicoccolide A is more straightforwardly explained starting from the precursor molecule flavipin, the presence of which as a primary building block was supported by HPLC–MS traces detected in crude extracts. The process begins with a thiamine-catalyzed unsymmetrical benzoin condensation involving the coupling of two aromatic moieties, each containing an aldehyde group, to form an intermediate compound, the unsymmetrical benzoin derivative flavipin. Following this condensation, the intermediate undergoes acetal formation, which is a critical step in the formation of the complex oxygen-bridged structure of the molecule. Final assembly is achieved by cyclization, in which ring closure is presumably mediated by benzaldehyde lyase.

Elucidation of these principles of natural assembly not only elucidates the structural relationships within the integrastatin family but also provides a clear biomimetic roadmap for the total synthesis strategies detailed in the following sections, particularly in overcoming the challenges of constructing the epoxybenzooxocine core.

4.2. Historical Multistep Total Syntheses

The first total synthesis of the tetracyclic core of integrastatin was reported by Foot and coworkers in 2003. Their approach utilized a Ramberg–Bäcklund reaction as the key transformative step (Scheme ).

3. Formation of the Tetracycle 7 through Ramberg–Bäcklund Reaction Precursor .

The synthesis commenced with the preparation of intermediates 6 and 7 from commercially available 2-methylacetophenone and 2-hydroxyacetophenone. The resulting thioether 8 was subsequently oxidized with m-chloroperbenzoic acid (m-CPBA) to give the corresponding sulfone 9. Following this, a chlorination-elimination sequence provided olefin 10, establishing the central unsaturated framework of the target molecule. In subsequent attempts to convert 10 to 11 using a Lewis acid, tetracycle 12 was observed in trace amounts as a side product. Tetracycle 12 was isolated as colorless crystals in 94% yield by intramolecular cyclization of the (Z)-isomer of compound 11 an excess of tin­(II) chloride dihydrate. To obtain the integrastatin core 13, tert-butyl hydroperoxide and pyridinium dichromate (PDC) were employedused (Scheme ).

4. Completion of the Integrastatin Core: Synthesis of 6,12-Dimethyl-6,12-epoxydibenzo­[b,f]­oxocin-11-one (13) .

In summary, the multistep synthesis of the integrastatin core by Foot and coworkers, featuring a Ramberg–Bäcklund reaction and Lewis acid-mediated cyclization, was achieved in 11 steps with an overall yield of 33%. While this strategy offered high regioselectivity and successfully established the complex tetracyclic framework, its practical application is constrained by a lengthy synthetic sequence, limited scalability, and the employment of toxic reagents.

Later, in 2005, Foot and coworkers investigated the effect of substituents within the sulfone system. It was demonstrated that replacing the ketal moiety with a keto group (14) or a hydroxyl group (15) led to a significant increase in the formation of the cis-product (16) during the Ramberg–Bäcklund reaction (Scheme ).

5. Effect of Functional Group Modifications in the Sulfone Precursor (14–15) on the Stereoselectivity of the Ramberg–Bäcklund Reaction and Subsequent Synthesis of the Integrastatin Nucleus (18) .

It was further demonstrated that only the cis-isomer 16 undergoes Lewis acid-catalyzed cyclization, which is believed to proceed by utilizing the olefin’s double bond to hold the starting olefin in a reactive conformation (impossible for the trans-isomer) to form the tetracycle 17. The structure of the integrastatin nucleus 18 has been ascertained through X-ray analysis of the crystalline product (Figure ). Furthermore, it was noted that the presence of a more electronegative nitro group in the same position did not provide the expected high yield of the Z-product. Similarly, the introduction of a methoxy substituent into the second aryl ring had no significant effect on the yield of the cis-isomer. Taken together, while this methodology provides unambiguous stereochemical control during cyclization, its synthetic scope remains constrained by substrate-dependent geometric requirements (cis-selectivity) and poor tolerance toward strongly electron-withdrawing substituents.

4.

ORTEP drawing of the Integrastatin nucleus (18). Reproduced from ref. with permission from the Royal Society of Chemistry.

Seeking a more expeditious route to the tetracyclic framework, in 2008 Ramana and coworkers reported a remarkably concise one-step entry to the integrastatin core. Their strategy was inspired by a retrosynthetic analysis of integrastatin B, which revealed that after an oxidation-state adjustment at C(9), the central core could be viewed as a pinacol cross-dimer of an o-ketoaldehyde (Scheme ). To test the feasibility of this biomimetic transformation, the authors employed commercially available o-phthalaldehyde (19) and o-hydroxybenzaldehyde (20) under various pinacol coupling conditions (Table ).

6. One-Step Construction of the Integrastatin Core via Pinacol Cross-Coupling of o-Phthalaldehyde and o-Hydroxy-benzaldehyde .

2. Reaction Conditions for the Pinacol Coupling of o-Phthalaldehyde (19) and o-Hydroxybenzaldehyde (20) (Scheme ) .

No.	Conditions	Yield
1	15% aq. solution of TiCl3, acetone, r.t.	7%
2	Zn, TiCl4, THF, 0 °C	21%
3	Mg(Hg), TiCl4, THF, 0 °C	42%
4	cat. Cp2TiCl2, Zn, TMSCl, THF, r.t.	15%
5	Mg, TMSCl, Cat InCl3, THF, r.t.	No reaction

It was found that the reaction was highly effective when using low-valent titanium reagents generated in situ. While reagents such as Zn–Cu or Zn provided the desired transformation, the best results were achieved using Mg­(Hg). This approach is particularly noteworthy for its efficiency, as it involves the consecutive formation of three chemical bonds in a single operation, directly affording the topologically complex tetracyclic system from simple precursors. Overall, while this methodology offers an unprecedented one-pot assembly of the core scaffold, its scope remains limited by the use of hazardous reducing agents (Mg­(Hg) and low-valent Ti), potential homocoupling side reactions, and restricted substrate compatibility.

Another method for the synthesis of the integrastatin core is based on the Grignard reaction between ketone 22 and benzyl bromide 23, as reported by Tadross and coworkers in 2011 (Scheme ).

7. Short-Step Synthesis of the Integrastatin Tetracyclic Core via Grignard Addition and Pd-Catalyzed Oxidative Cyclization .

To remove the protecting tert-butyldimethylsilyl (TBS) group, the resulting tertiary alcohol 24 was treated with tetrabutylammonium fluoride (TBAF). Subsequent treatment of diol 25 with catalytic amounts of PdCl₂ and CuCl₂ under an oxygen atmosphere induced cyclization. Thus, the synthesis of the tetracyclic core 18 was achieved in only 4 steps from known compounds, with an overall yield of 30%. In essence, this methodology provides a highly efficient and step-economical entry into the core scaffold using catalytic oxidative cyclization. However, its broader application is limited by the strict requirement for protecting-group manipulation (TBS removal) and the operational sensitivity of Grignard additions toward functional groups.

The first total synthesis of integrastatin B was achieved in 7 steps with an overall yield of 17.9% by More and Ramana in 2016 (Scheme ).

8. Total Synthesis of Integrastatin B via a Benzofuran Oxidative Dearomatization Cascade .

The key stage in this scheme is the synthesis of benzofuran 28, which was isolated via a Fürstner–McMurry reaction from ester 27. This ester was prepared by reacting 2-hydroxy-3,4-dimethoxy-6-methylacetophenone 26 with commercially available 3,4,5-trimethoxybenzoic acid (eudesmic acid). Subsequently, benzofuran 28 underwent Friedel–Crafts acetylation to afford the 6-methyl-substituted compound 29. This was followed by the selective hydrolysis of three of the five methoxy groups with boron trichloride. Subsequent acylation of the three resulting phenolic groups yielded compound 31, which underwent a skeletal reorganization into tetracycle 32 upon treatment with potassium peroxymonosulfate. Finally, selective oxidation of the arylmethyl group to the corresponding aldehyde, followed by the hydrolysis of the acetoxy groups, completed the synthesis. Overall, while this strategy provided an efficient entry into the natural product via a landmark biomimetic-type cascade, its practical scalability is hampered by lengthy functional-group manipulations, strict requirements for selective methoxy cleavage, and low yield in the final oxidation sequence.

Next, More and Ramana, in 2016, developed a more general and mechanistically sophisticated approach to the [6,6,6,6]-tetracyclic core common to integrastatins, epicoccolide A, and epicocconigrone A:

This strategy centers on generating transient o-quinone methides (o-QMs) via oxidative dearomatization of C2-arylbenzofuran precursors (Scheme ).

9. Synthesis of Benzofuran 2a and Its Oxidative Dearomatization Cascade to the Integrastatin Core .

ⁱ Adapted and reproduced from ref. with permission from the Royal Society of Chemistry.

The authors demonstrated that treatment of these benzofurans with oxone in an acetone–water mixture at room temperature efficiently triggers the dearomatization process. The resulting highly reactive o-QM intermediates (33) undergo a spontaneous intramolecular [4 + 2] cycloaddition with a tethered carbonyl group (or a conjugated olefin) located on the C2-aryl moiety. This cascade sequence allows for the expeditious construction of the oxygen-bridged tetracyclic framework in a single operational step, providing a powerful biomimetic tool for accessing this rare class of natural products. The principal advantages of this methodology lie in its high step economy, mild reaction conditions, and broad versatility for accessing related natural scaffolds. However, its main limitations involve the operational challenge of managing highly reactive, short-lived o-QM intermediates, potential competing oxidative side-reactions, and a dependency on specific substituent positioning to ensure favorable geometry for the intramolecular [4 + 2] cycloaddition.

The most recent advance in the construction of the integrastatin quaternary framework is the work reported in 2019 by Jeong (Scheme ).

10. Synthesis of the Integrastatin Core via Aryllithium Coupling and pTSA-Mediated Acid-Catalyzed Cyclization .

As illustrated in Scheme , the synthesis commenced with the protection of commercially available 2-bromophenol (34) as its ether derivative 35. Alkylation of the resulting bromide 35 with methyl pyruvate afforded the hydroxy ester 36. Subsequent reduction of ester 37 yielded aldehyde 38 in 74% overall yield for the two steps, and this aldehyde then underwent a coupling reaction with an aryllithium species.

The coupling product 39 was successfully obtained in 83% yield. The newly formed configuration at C-1 was removed in a subsequent oxidation step to provide the cyclization precursor 40. Finally, subjecting compound 40 to harsh deprotection conditions, followed by cyclization using pTSA·H₂O afforded the tetracyclic integrastatin core 41. This methodology enables the construction of the integrastatin core in 8 steps, providing high reaction yields throughout the sequence. Thus, while this route offers high chemical efficiency and a dependable yield profile across all intermediate transformations, its scalability remains constrained by the necessity of organometallic intermediates and rigorous deprotection steps that require aggressive acidic conditions.

A significant improvement in the synthetic accessibility of the integrastatin framework was recently reported by Yamagiwa and coworkers in 2020, who developed a highly versatile and practical route to the tetracyclic core. By utilizing o-toluic acid and 2’-hydroxyacetophenone with NaH/sec-BuLi, followed by the reaction of dihydroisocoumarins with MeLi, the authors successfully streamlined the process to only 3 steps (Scheme ).

11. Improved 3-Step Synthesis of the Integrastatin Core via Dihydroisocoumarin Intermediates .

By employing a dianion methodology according to Kessar and Beam, followed by acetal formation with methyllithium, this approach significantly shortened the synthetic route to model compound 18. An investigation into the scope and limitations of the substrate revealed that the presence of various substituents on the aromatic ring is well tolerated, generally affording an acceptable product yield of 9%. Consequently, while this route offers an unprecedentedly short 3-step access to the tetracyclic core from simple building blocks, its practical utility is constrained by a modest overall yield and the operational hazards associated with reactive organolithium dianion intermediates.

Overall, the construction of the [6,6,6,6]-tetracyclic core of integrastatins has evolved significantly since the first reports, reflecting broader trends in modern organic synthesis toward step efficiency. Whereas early strategies, such as those by Foot, relied on classical functional group transformations and multistep sequences, subsequent methodologies have increasingly utilized powerful cascade reactions and metal-mediated couplings. Notable advancements include the remarkably concise one-step pinacol coupling developed by Ramana, as well as the sophisticated oxidative dearomatization protocols that allow for the spontaneous assembly of the oxygen-bridged framework. While these methods established the feasibility of core construction, they often suffered from low yields and required expensive reagents.

Despite these breakthroughs, the synthesis of heteroatom-substituted variants, particularly those incorporating pyridine and pyrimidine moieties, remains a formidable challenge because of the electronic divergence of these heterocycles compared to their carbocyclic counterparts. Addressing these limitations is critical for the development of potent HIV-1 integrase inhibitors with improved pharmacokinetic profiles. Consequently, the following sections will focus on recent one-step strategies for pyridine and pyrimidine analogues, highlighting modern methodologies that streamline the construction of these complex scaffolds while maintaining high atom economy and functional group tolerance.

4.3. Recent One-Step Strategies for Pyridine and Pyrimidine Analogs

In 2021, a simple, one-step synthetic route to pyridine-containing integrastatin analogs 44 was reported by Kulakov et al. This strategy involves the acid-catalyzed cyclization of 3,5-diacetyl-2,6-dimethylpyridine 42 with various salicylic aldehyde derivatives 43 into epoxybenzooxocines 44 followed by oxidation to a carbonyl moiety (Scheme ).

12. Acid-Catalyzed Intramolecular Cyclization into Epoxybenzooxocines and Their Subsequent Oxidation to the Carbonyl Moiety .

This acid-catalyzed cyclization demonstrates broad substrate tolerance toward both electron-withdrawing and electron-donating groups, offering a versatile platform for the synthesis of diverse nitrogen-containing integrastatin analogues (Figure ). To evaluate the versatility of the optimized protocol, the authors , investigated the substrate scope using a diverse set of substituted salicylaldehydes bearing both electron-donating and electron-withdrawing groups (Scheme ). The cyclization was performed under reflux in 2-propanol or butanol with 10 mol % trifluoroacetic acid, facilitating the precipitation of 21 new oxocine derivatives, 44a–u, as crystalline solids.

5.

ORTEP representations of the molecular structure of compound 44a.

13. Synthesis of 11,12-Dihydro-5H-5,11-epoxybenzo­[7,8]­oxocino­[4,3-b]­pyridine Derivatives 44a–u .

It was shown that the electronic nature of the substituents significantly influenced the reaction kinetics, with acceptor groups accelerating the transformation while donor-substituted substrates required higher temperatures in sealed ampules to achieve optimal yields. The structures of the resulting tetracyclic scaffolds were unambiguously confirmed by NMR spectroscopy and single-crystal X-ray diffraction data (Figure ), in which the characteristic signals of the diastereotopic methylene protons and the epoxybenzooxocine methine proton served as definitive diagnostic markers.

A subsequent in silico study of the physicochemical and bioactivity parameters of this series of 21 new synthetic analogues of natural integrastatins by Pustolaikina et al. was conducted. Using the online resource PASSOnline, the antiviral activity and CYP2C19 inhibitory potential of compounds 44a–u inducers were demonstrated. Molecular docking results showed that compound 44u exhibited high antiviral potential against HIV-1 (PDB ID 3V81) and SARS-CoV-2 (PDB ID 7AAP). An assessment of the ADMET properties using the ADMETlab 3.0 online portal revealed the potential for high oral efficacy of the studied compounds and a promising safety profile for their therapeutic use.

The library of integrastatin analogues was further expanded in 2023 via a comprehensive experimental and computational study on the cyclization of various 3-acetyl-2-methylpyridines and quinolines with salicylaldehyde (Scheme ).

14. Cyclization of Various 3-Acetyl-2-methylpyridines and Quinolines with Salicylaldehyde .

The reactivity of the pyridine-based precursors depended strongly on their electronic and steric profiles, with electron-donating groups facilitating cyclization by enhancing the C-H acidity of the ortho-methyl group through nitrogen protonation (Table ).

3. A Study on the Cyclization of Diverse 3-Acetyl-2-methylpyridines and Quinolines with Salicylaldehyde .

The authors demonstrated that while electron-donating substituents significantly facilitate the formation of the epoxybenzooxocino­[4,3-b]­pyridine skeleton, the presence of electron-withdrawing groups, such as a 5-nitro group, requires more rigorous conditions in sealed ampules to achieve high conversion. Notably, the study utilized quantum chemical calculations to elucidate the lack of reactivity in 4-aryl-substituted derivatives, which was attributed to significant steric hindrance that prevents effective cyclization. Furthermore, the demonstrated ability of specific pyridine derivatives to undergo secondary cyclizations into complex regioisomeric and diastereomeric bisoxocines highlights the potential of this methodology for constructing highly sophisticated, bioinspired architectures.

In 2024, a straightforward one-step protocol for the synthesis of 5-methyl-11,12-dihydro-5H-5,11-epoxybenzo­[7,8]­oxocino­[4,3-d]­pyrimidine derivatives was reported, which utilized the acid-catalyzed cyclization of substituted 4-methyl-5-acetylpyrimidines with salicylaldehyde (Scheme ).

15. One-Step Synthesis of EpoxybenzooxocinoPyrimidine Derivatives via Acid-Catalyzed Cyclization of Pyrimidines with Salicylaldehyde .

The reaction was shown to proceed smoothly for 2-substituted 4-methyl-5-acetylpyrimidines, affording the corresponding cyclized products 60a–e. Conversely, the introduction of a substituent at the C-6 position of the pyrimidine ring completely suppressed the cyclization. This reactivity limitation is attributed to steric hindrance caused by C-6 substituents, which restricts the conformational rotation of the acetyl group and precludes the assembly of the necessary prereaction complex. The molecular structures of the target 5-methyl-11,12-dihydro-5H-5,11-epoxybenzo­[7,8]­oxocino­[4,3-d]­pyrimidines (60a–e) were unambiguously confirmed by ¹H and ¹³C NMR spectroscopy, mass spectrometry, and single-crystal X-ray diffraction analysis (Figure ).

6.

Single-crystal X-ray structure (ORTEP diagram at 50% probability level) of derivative 60c.

Overall, acid-catalyzed one-step cyclizations of acetyl- and methyl-substituted pyridines, quinolines, and pyrimidines with salicylaldehydes provided access to nitrogen-fused integrastatin analogues. The reaction scope is governed by electronic and steric factors, with C-6 substitution on the pyrimidine ring suppressing reactivity. Structural characterization by single-crystal X-ray diffraction confirms the formation of these fused systems, while in silico evaluations indicate potential antiviral activity against HIV-1 and SARS-CoV-2. These transformations establish a direct synthetic route to functionalized epoxybenzooxocine derivatives.

4.4. Total Synthesis of Epicoccolides A and B by Oxidative Skeleton Rearrangement

A landmark total synthesis of epicoccolides A and B was achieved by Treiber and coworkers in 2025, employing a modular strategy centered on a PtCl₂-catalyzed alkyne cyclization and a subsequent oxidative skeleton rearrangement (Scheme ).

16. Total Synthesis of Epicoccolide B (5) .

The total synthesis of Epicoccolide B (5) was achieved through a strategic 13-step linear sequence, starting from the common precursor 61. The initial phase involved the quantitative conversion of an ester to the aldehyde 62 via a reduction–reoxidation protocol, followed by a highly efficient electrophilic aromatic iodination to yield fragment 63. This intermediate served as a pivotal branching point, providing access to the second key fragment, vinyl-alkyne 64, through an Ohira–Bestmann homologation and subsequent Suzuki–Miyaura cross-coupling.

The convergent assembly of the molecular framework was realized via a Sonogashira coupling between fragments 63 and 64, where the presence of the vinyl group proved to be essential for achieving high conversion levels. Following oxidative cleavage of the terminal olefin and global deprotection to liberate the free phenol 67, the synthesis culminated in a signature-catalyzed alkyne cyclization. This biomimicry-like ring closure successfully afforded Epicoccolide B (5) in a total yield of 10%, with all spectroscopic data matching the natural metabolite and confirming its complex aromatic polyketide architecture.

Having successfully completed the total synthesis of Epicoccolide B (5), the authors turned their attention to validating the proposed biomimetic pathway through an oxidative skeletal rearrangement (Scheme ).

17. Oxidative Skeleton Rearrangement of Epicoccolide B (5) to Epicoccolide A (3) and Structural Reassignment toward Epicocconigrone A (4) .

This proposed biomimetic sequence, proceeding via initial furan oxidation to intermediate 68, followed by a sequential [4 + 2] cycloreversion–cycloaddition of species 69, logically accounts for the formation of the Epicoccolide A framework as the final rearrangement product (Scheme ). The structural constitution of compound 3 was unambiguously confirmed through comprehensive 2D NMR spectroscopy, where key HMBC correlationsspecifically from H-17 to C-12, H-18 to C-11, and from H-1, H-9, and H-16 to C-7established the connectivity of the tetracyclic framework. These findings were further supported by NOE correlations between H-17/H-18 and the aldehyde proton/H-9, providing definitive proof of the spatial arrangement. Ultimately, Treiber and coworkers in 2025 showed, through careful analysis, that the NMR data for the originally proposed structure of Epicoccolide A were identical to those published for Epicocconigron A (4) by Proksch’s group in 2013. Based on this, Treiber and coworkers concluded that the material previously isolated by Laatsch and colleagues was, in fact, Epicocconigron A, thus correcting a long-standing structural misinterpretation in the literature.

In conclusion, the authors reported the first total synthesis of epicoccolide A (3), epicoccolide B (5), and epicocconigrone A (4), relying on two different routes, starting from a common precursor, to synthesize the benzofuran motif. The routes are scalable and proceed with high yields. Key steps involve an Ullmann-type coupling, an alkyne ring cyclization, and a novel air oxidation of unprotected epicoccolide B, which can be reproduced under controlled conditions using DMDO.

Overall, to sum it up, the synthetic journey from the first isolation of epicoccolides to the definitive total synthesis by Treiber et al. underscores the indispensable role of total synthesis in structural validation. The implementation of a modular PtCl₂-catalyzed cyclization, followed by a biomimetic oxidative rearrangement, not only provided a robust chemical route to these metabolites but also served as a rigorous analytical tool. The subsequent structural reassignment of epicoccolide A to epicocconigrone A highlights the challenges inherent in the characterization of complex polysubstituted aromatic polyketides and the necessity of high-field 2D NMR techniques (HMBC and NOE) in resolving structural ambiguities.

4.5. Comparative Analysis and Evaluation of Synthetic Strategies for the Epoxybenzooxocine Core

An analysis of the literature data from 2003 to 2025, reviewed above, reveals a clear evolutionary trajectory: from classical multistep syntheses to biomimetic cascade processes and, ultimately, to one-step acid-catalyzed methods. To objectively evaluate the efficiency of these synthetic approaches, a comparative analysis was performed across key parameters, including step count, overall yield, reagent availability, scalability, access to heterocyclic analogues, and biomimetic design (Table ).

4. Comparative Analysis of Synthetic Strategies toward the Epoxybenzooxocine Core.

Authors (Year)	Key Transformation	Number of Steps	Overall Yield, %	Reagent Availability	Scalability	Heterocycles	Biomimetic Design
Foot et al. (2003, 2005) ,	Ramberg–Bäcklund + SnCl2	11	33	Moderate	Low	-	-
Ramana et al. (2008)	Pinacol cross-coupling (Ti)	1	up to 42	Low (Hg, Ti)	Low	-	+
Tadross et al. (2011)	Grignard + Pd- catalyzed cyclization	4	30	Low (Pd)	Moderate	-	-
More and Ramana (2016) ,	Oxone-mediated dearomatization	7	18	Moderate	Low	-	+
Jeong et al. (2019)	Aryllithium + pTSA	8	High	Low (sec-BuLi)	Low	-	-
Yamagiwa et al. (2020)	Dianion addition + MeLi	3	9	High	Low	-	-
Kulakov et al. (2021–2024) −	TFA catalysis, pyridines/pyrimidines + salicylic aldehydes	1	Varies	High	High	+	+
Treiber et al. (2025)	PtCl2 + oxidative rearrangement	13	10	Low (Pt)	Low	-	+

^aHigh yields were reported for individual steps; the overall yield was not specified by the authors.

^bDepending on the nature of the substituents, electron-donating groups afford higher yields, whereas electron-withdrawing groups require harsher conditions.

The data presented in Table indicate that early strategies, including Foot’s , synthesis via the Ramberg–Bäcklund reaction (11 steps, 33% overall yield), established the basis for constructing the target scaffold but suffered from limited scalability and the use of toxic reagents. Subsequent routes, such as Ramana’s pinacol cross-coupling (1 step, up to 42% yield), the Tadross method (4 steps, 30%), and More and Ramana’s cascade oxidative dearomatization (7 steps, 18%), , demonstrated the clear benefits of biomimetic design, yet they did not address the synthesis of heterocyclic analogues. Synthetic strategies, particularly the total synthesis by Treiber et al., have underscored the critical role of organic synthesis not only as a tool for target molecule assembly, but also as a definitive means for the structural validation of natural metabolites. The structural reassignment of epicoccolide A to epicoccogyrone A based on total synthesis data highlights the necessity of combining high-field 2D NMR spectroscopy (HMBC, NOESY) with chemical synthesis for the unambiguous structural elucidation of complex polysubstituted aromatic polyketides.

Based on the data summarized in Table , the reviewed synthetic methods can be categorized by key efficiency criteria (Table ).

5. Categorizing of Synthetic Methods − by Key Efficiency Criteria.

Criterion	Most Efficient Method	Remarks
Minimum number of steps	Ramana (1 step), Kulakov (1 step)	Both methods afford the target scaffold in a single operational step
Maximum overall yield	Foot (33%), Tadross (30%)	Classical approaches deliver the highest cumulative yields
Reagent availability	Yamagiwa, Kulakov	Do not require precious transition metals or specialized reagents
Scalability	Tadross, Kulakov	Straightforward reaction conditions and simple product isolation
Access to fused heterocycles	Kulakov	All other methods are restricted to carbocyclic cores
Biomimetic design	Ramana, More–Ramana, Treiber	Most closely emulate proposed biosynthetic pathways

The data presented in Table indicate that a minimum step count (1 step) is achieved by only two approaches, namely Ramana’s pinacol coupling and Kulakov’s acid-catalyzed cyclization. However, while Ramana’s approach yields only carbocyclic cores and relies on hazardous reagents, the Kulakov method offers direct access to heterocyclic analogues. Although the highest overall yields (30–33%) are delivered by the Foot and Tadross protocols, they necessitate 11 and 4 synthetic steps, respectively. In terms of reagent availability and scalability, protocols relying on expensive transition metals (such as Pd, Pt, Ti, and Hg) or hazardous reagents, such as those reported by Foot, Ramana, Jeong, Yamagiwa, and Treiber, exhibit limited scalability. In contrast, Kulakov’s acid-catalyzed protocol avoids costly reagents, rendering it preferable for preparative-scale synthesis. The most biomimetic strategies include Ramana’s method (reductive aldehyde cross-coupling), More and Ramana’s protocol (oxidative dearomatization), and Treiber’s approach (oxidative skeletal rearrangement). Although not a direct biomimetic reproduction of the natural pathway, the Kulakov method employs principles of acid catalysis analogous to enzymatic processes. This strategy, based on the acid-catalyzed cyclization of 3-acetyl-2-methylpyridines, quinolines, and pyrimidines with salicylaldehydes, − provides a direct one-step route to nitrogen-containing heterocyclic integrastatin analogues that cannot be realized by other synthetic routes. Overall, the acid-catalyzed method simultaneously satisfies three key criteria: minimal step count, high scalability, and direct access to fused heterocyclic analogues of integrastatins. This makes it the most promising strategy for constructing compound libraries with predicted activity against HIV-1 and SARS-CoV-2. Nevertheless, the method has its limitations: electron-withdrawing substituents require harsher conditions (sealed tubes), while steric hindrance, particularly in the pyrimidine series, can completely suppress the reaction.

In summary, the synthetic chemistry of the epoxybenzooxocine core has evolved from initial proof-of-concept constructions of this scaffold to operationally simple, single-step protocols that afford access to a broad array of heterocyclic analogues of natural integrastatins. The strategy, based on the acid-catalyzed cyclization of 3-acetyl-2-methylpyridines, quinolines, and pyrimidines with salicylaldehydes, currently offers the most viable framework for generating focused libraries of potential antiviral agents and subsequently optimizing their pharmacological profiles. Despite this progress, several key challenges remain, including improving the overall yield of the acid-catalyzed method (particularly for substrates with electron-withdrawing substituents), expanding the substrate scope to other nitrogen-containing heterocycles (e.g., pyrazines, cinnolines, quinoxalines, triazines), and performing comprehensive biological screening of the synthesized analogues to confirm their predicted in silico activity and optimize their structures.

5. Epoxybenzooxocine Scaffolds: Structure, Pharmacophoric Insights, and ADMET Profiles

Analysis of the synthetic approaches and epoxybenzooxocine derivatives reviewed herein reveals three fundamental scaffolds that serve as core molecular frameworks: the unsubstituted natural core (scaffold 1), the pyridine-fused architecture (scaffold 2), and the pyrimidine-fused system (scaffold 3). These 3 molecular scaffolds represent rigid heterocyclic cores that provide a defined spatial foundation for the precise arrangement of R-group substituents. Furthermore, these core scaffolds directly dictate the baseline physicochemical and ADMET profiles of the resulting derivatives, including fundamental properties such as lipophilicity (log p), molecular volume, shape, and topological polar surface area (TPSA). By restricting random conformational rotation, these rigid frameworks minimize entropic penalties upon binding to enzymes or receptors while precisely orienting key pharmacophoric vectorssuch as −OH, −NO₂, −NH₂, and aromatic ringsat optimal distances and angles to ensure geometric and electronic complementarity with the target binding pocket (Figure ).

7.

Comparative 2D SAR mapping and pharmacophoric feature distribution across the natural epoxybenzooxocine core (1) Scaffold 1, its synthetic pyridine-fused Scaffold 2, and pyrimidine-fused Scaffold 3. Color coding highlights specific pharmacophoric vectors: green = hydrophobic and halogen features (−CH₃, −C₂H₅, −Br); red = hydrogen-bond acceptors (−CHO, −NO₂, −COCH₃, −OCH₃); cyan = hydrogen-bond donors (−OH, −NH₂); yellow = aromatic/hydrophobic extensions (phenyl ring).

The structural evolution of epoxybenzooxocine-based architecturesfrom the natural integrastatin core (Scaffold 1, discovered in 2002), through the epoxybenzooxocinopyridine framework (Scaffold 2, reported in 2021), to the epoxybenzooxocinopyrimidine system (Scaffold 3, published in 2024)represents a strategic optimization of both pharmacophoric complexity and synthetic efficiency.

The natural epoxybenzooxocine framework (1) features a fully carbocyclic, fused tetracyclic core combined with multiple oxygenated functional groups and alkyl substituents distributed across both benzene rings (R₁–R₈). It provides a polyfunctionalized interaction network containing multiple hydrogen-bond donor (HBD, −OH) and hydrogen-bond acceptor (HBA, −CHO, −OCH₃) vectors alongside hydrophobic methyl groups (−CH₃). Despite its potent baseline antiviral biological activity, the synthesis of substituted natural analogues requires multistep procedures, specific reaction conditions, and often results in low product yields, while the presence of sensitive groups limits metabolic stability and precise SAR tuning.

Replacement of one benzene ring of the natural epoxybenzooxocine core in Scaffold 1 with an annulated pyridine heterocycle in Scaffold 2 results in simplified synthetic access and increased basicity. The endocyclic pyridine nitrogen acts as a fixed hydrogen-bond acceptor and a potential protonation center, thereby modulating solubility and target binding. Functionalization at the pyridine positions R₁-R₃ introduces strong electron-withdrawing HBA groups (−NO₂, −COCH₃), alkyl chains (−CH₃, −C₂H₅), and aromatic/hydrophobic extensions (phenyl rings). The remaining benzene moiety R₄-R₇ accommodates dual-character HBA/halogen substituents (−Br) alongside classic HBD (−OH) and HBA (−OCH₃) vectors. This step enables high-yielding, one-pot, acid-catalyzed cyclizations from readily available 3-acetylpyridines and salicylaldehydes.

Further evolutionary refinement led to the introduction of a pyrimidine ring (scaffold 3), creating a diazine-fused epoxybenzooxocine topology. In contrast to scaffolds 1 and 2, which were established earlier and have been extensively explored through multipositional modifications, scaffold 3 was introduced in 2024 and is currently represented by structural variations concentrated at a single, highly accessible position (C-2 of the pyrimidine ring, vector R). The C-2 position tolerates a diverse range of functional groups, including pure HBD (−NH₂), hydrophobic (−OCH₃), HBA/hydrophobic (−OCH₃), and aromatic (phenyl) moieties. The unsubstituted C-6 position on the pyrimidine ring is essential; introduction of substituents at C-6 completely suppresses cyclization due to steric hindrance, preventing acetyl rotation. Scaffold 3 provides maximum pharmacophoric versatility with minimal synthetic complexity, establishing a highly focused platform for structure–activity relationship (SAR) fine-tuning and SBDD (structure-based drug design).

Consequently, it was of particular interest to trace how key physicochemical parameters and ADMET profiles evolve across the structural transition from Scaffold 1 to Scaffold 3. To transition from structural evaluation to pharmacokinetic characterization, a representative compound was selected from each family (2a for Scaffold 1, 44a for Scaffold 2, and 60b for Scaffold 3) to evaluate and compare their overall ADMET profiles, physicochemical properties, and drug-likeness parameters using predictive in silico modeling. The calculated by AdmetLab3 online tool (https://admetlab3.scbdd.com/server/) physicochemical drug-likeness profiles and in silico ADMET parameters for Scaffolds 1–3 and compounds 2a, 44a, and 60b are summarized in Figure and Table , respectively.

8.

Chemical structures and radar charts of the physicochemical profiles (drug-likeness) of Scaffolds 1–3 and compounds 2a, 44a, and 60b.

6. Predicted In Silico ADMET Profiles of Scaffolds 1–3 and Compounds 2a, 44a, and 60b .

^aAll values were calculated using ADMETlab 3.0. Caco2 and MDCK permeability (optimal: higher than −5.15 log units). HIA (P < 0.3 indicates high absorption). Blood–brain barrier (BBB) penetration probability (<0.3 indicates nonpenetrant). Plasma protein binding (PPB) percentage (optimal: <90%). Probability of CYP2C19 inhibition (Category 1: Inhibitor; Category 0: Noninhibitor). Human liver microsomal (HLM) stability score (<0.5 indicates potential instability). CL Plasma clearance (>15 mL/min/kg: high clearance; 5–15 mL/min/kg: moderate clearance; <5 mL/min/kg: low clearance). T _1/2 Elimination half-life (ultrashort half-life drugs: T _1/2 < 1 h; short half-life drugs: T _1/2 between 1 and 4 h; intermediate short half-life drugs: T _1/2 between 4 and 8 h; long half-life drugs: T _1/2 > 8 h). Probability of hERG potassium channel blockage (molecules with IC₅₀ ≤ 10 μM or ≥50% inhibition at 10 μM were classified as hERG+ Category 1, while molecules with IC50 > 10 μM or <50% inhibition at 10 μM were classified as hERGCategory). Human hepatotoxicity probability (Category 1: H-HT positive­(+); Category 0: H-HT negative(−)). Carcinogenicity probability (Category 1: carcinogens; Category 0: noncarcinogens). Skin sensitization probability (Category 1: Sensitizer; Category 0: Nonsensitizer). Respiratory toxicity probability (Category 1: respiratory toxicants; Category 0: nonrespiratory toxicants).

^bColor coding according to ADMETlab 3.0 evaluation criteria: a green circle indicates a favorable/optimal parameter value (low risk); a yellow circle represents a moderate/medium risk parameter; and a red circle denotes an unfavorable/suboptimal parameter value (high risk).

Analysis of the physicochemical radar charts reveal that the unsubstituted epoxybenzooxocine cores (Scaffolds 1–3) possess balanced drug-likeness profiles, with all key descriptors falling within the optimal boundaries (shaded blue area, Figure ). Structural modification of these cores to yield derivatives 2a, 44a, and 60b induces predictable shifts in physicochemical space. Specifically, polyhydroxylation and methoxylation in 2a increase molecular weight (MW) and topological polar surface area (TPSA) relative to parent Scaffold 1. Similarly, heteroaromatic fusion and functionalization in 44a and 60b modulate hydrogen bond acceptor (nHA) counts and rotatable bonds while retaining favorable lipophilicity and structural rigidity. Overall, all evaluated derivatives preserve high compliance with standard drug-likeness criteria, validating the epoxybenzooxocine scaffold as a lead-like chemical framework for further optimization.

The in silico ADMET evaluation (Table ) provides insights into the pharmacokinetic profiles and safety liabilities of Scaffolds 1–3 and their representative derivatives (2a, 44a, and 60b). All compounds demonstrate good predicted human intestinal absorption (HIA) along with a low risk of cardiotoxicity, as evidenced by favorable hERG inhibition probabilities (<0.33). Parent Scaffolds 1–3 and derivatives 44a and 60b generally share a profile marked by high blood–brain barrier penetration (BBB > 0.88), strong plasma protein binding (PPB > 90%), and potential CYP2C19 inhibition. In contrast, derivative 2a displays a distinct pharmacokinetic signature, featuring restricted central nervous system permeability (BBB = 0.014), a complete lack of CYP2C19 inhibition (0.000), and significantly lower respiratory toxicity (0.089). The reduced risk profile and restricted permeability of compound 2a (integrastatin A) are likely driven by its extensive functionalization with multiple substituents R_1,2,4,6,7,8, whereas Scaffolds 1–3 and derivatives 44a and 60b possess far fewer substituents, which accounts for their significantly higher membrane permeability

Overall, although the core framework offers favorable absorption and cardiac safety, the predicted liabilitiesparticularly elevated human hepatotoxicity (H-HT), carcinogenicity, and skin sensitization probabilities across the serieshighlight key areas that must be addressed through targeted structural modifications in the future.

6. Functionalization, Biological Screening, and Computational Profiling of Epoxybenzooxocine Derivatives

After establishing robust synthetic models for constructing the epoxybenzoxocine core, the researchers’ primary focus shifted to postsynthetic modifications and functionalization strategies required to obtain a broader library of derivatives with tailored physicochemical properties. Using compound 44a (Scaffold 2) as a core framework, over 30 novel epoxybenzooxocine derivatives were synthesized between 2022 and 2025 through targeted substitutions at the C-3 position of the pyridine ring − and at the C-11 position of the fused benzene moiety.

Scheme illustrates the systematic functionalization of the epoxybenzooxocinopyridine core derivative 44a (Scaffold 2), at two primary reactivity centers: the acetyl group at the C-3 position of the pyridine ring and the fused benzene ring at the C-11.

18. Structural Diversification of Derivative 44a (Scaffold 2) via Functionalization at the C-3 (Pyridine) and C-11 (Benzene) Positions ,,,

Modifications at the C-3 position yielded a diverse library of structural analogues, including hydrazones (70–71), β-dicarbonyl derivatives (72, 75), quinoxalin-2-one conjugates (73a–d), oximes (74a–b), pyrazoles (76a–b, 77a–b), 1,4-dihydropyridines (78, 79), secondary alcohol (80), pyridylchalcones (81a–e), and 1-acetyl-4,5-dihydro-1H-pyrazoles (82a–e).

Parallel functionalization at the C-11 position of the benzene moiety afforded derivatives 83a–i bearing S-methylthio, N-methylamino, heterocycle-linked, and polycyclic substituents. Overall, this dual-vector modification strategy enabled the synthesis of over 30 novel derivatives with expanded pharmacophoric complexity.

To assess the therapeutic potential of scaffold 2 derivatives against SARS-CoV-2, their cytotoxicity (IC₅₀) and antiviral activity (EC₅₀) were determined in Vero E6 cells using tilorone as a positive control (Table ).

7. Cytotoxicity and Antiviral Activity against SARS-CoV-2 in Vero E6 cells .

Compound	IC50, μg/mL	EC50, μg/Ml
44a	131.1	>200
70	>200	>200
71	>200	>200
72	>200	>200
73a	6.0	2.2
73b	>200	>200
73c	>200	>200
Tilorone	7.8	4.3

^aThe upper limit of concentrations tested is 200 μg/mL. If the value exceeds the upper limit of the tested concentrations, “>200” is indicated.

In vitro screening of the epoxybenzooxocinopyridine series identified compound 73a as the most potent candidate. Molecular docking against SARS-CoV-2 RNA-dependent RNA polymerase (RdRp, PDB ID: 7AAP) revealed significantly stronger binding affinity for 73a compared to tilorone (−9.7 vs −6.7 kcal/mol) (Figure ). Within the active site, 73a forms key interactions, including a metal–acceptor bond with Mg1003, a hydrogen bond with Asp623, π–cation interactions with Arg553 and Lys621, a π–alkyl contact with Arg624, and a π–π T-shaped interaction with Tyr455.

9.

Molecular docking of compound 73a with SARS-CoV-2 RNA-dependent RNA polymerase (PDB ID: 7AAP): 3D binding view (left) and corresponding 2D interaction diagram (right).

To evaluate the antimicrobial profile of Scaffold 2 derivatives (44a, 70–80), their minimum inhibitory concentrations (MICs) were determined against Mycobacterium bovis 14, Staphylococcus aureus, Pectobacterium carotovorum, and Candida albicans, with Isoniazid serving as the positive control (Table ).

8. Minimum Inhibitory Concentration (MIC) for the Antimicrobial Activity of epoxybenzo­[7,8]­oxocino­[4,3-b]­pyridine derivatives .

	MIC (μg/mL)
Compound	Mycobacterium bovis 14	Staphylococcus aureus 209 P	Pectobacterium carotovorum VKM-B1247	Candida albicans ATCC 10231
44a	50	>1000	>1000	>1000
70	0.78	>1000	>1000	>1000
71	12.5	500	>1000	1000
72	1.56	250	>1000	1000
73a	0.39	>1000	>1000	>1000
73b	0.39	>1000	>1000	>1000
73c	0.78	125	>1000	1000
73d	6.25	1000	>1000	>1000
74a	25	>1000	>1000	>1000
74b	25	>1000	>1000	>1000
75	0.39	125	>1000	125
76a	12.5	>1000	>1000	>1000
76b	50	1000	>1000	>1000
77a	12.5	>1000	>1000	>1000
77b	3.12	>1000	>1000	>1000
78	50	>1000	>1000	>1000
79	12.5	>1000	>1000	>1000
80	25	>1000	>1000	>1000
Isoniazid	0.195	–	–	–

Biological evaluation in ref. confirmed the antitubercular potential of epoxybenzo­[7,8]­oxocino­[4,3-b]­pyridines, with compounds 73a–73b and 75 exhibiting high activity against M. bovis (MIC down to 0.39). Molecular docking against six mycobacterial targetsthe C-type lectin mincle (PDB ID: 4KZW), adenosine kinase (PDB ID: 4UBE), 7,8-diaminopelargonic acid synthase (BioA) (PDB ID: 5TE2), cytochrome P450 enzyme 121 (PDB ID: 6TET), UDP-N-acetylmuramic acid l-alanine ligase (MurC) (PDB ID: 7BVB), and tryptophanyl-tRNA synthetase (PDB ID: 7ENS)using isoniazid as a comparator revealed high binding affinities, notably exceeding −10 kcal/mol for BioA (5TE2) and CYP121 (6TET). Most derivatives surpassed the comparator ligand across targets, except for CYP121, where only 73a and 73c demonstrated superior binding. The BioA-77b complex exhibited the lowest binding energy, stabilized by structural shape complementarity, four hydrogen bonds, and 12 hydrophobic (π-alkyl/alkyl) contacts with key active-site residues (Ala226, Gly159, Tyr157, Lys283, Ala257, and Ile256) (Figure ).

10.

Molecular interactions of 77b within the active site of 7,8-diaminopelargonic acid synthase (PDB ID: 5TE2): (Left) 3D views with aromatic and hydrogen-bond surface mappings; (Right) 2D interaction diagram.

The tuberculostatic potential of synthesized pyridylchalcones (81a–e) and 4,5-dihydro-1H-pyrazoles (82a–e) against Mycobacterium bovis was systematically evaluated across a broad concentration gradient (0.195–100 μg/mL) using isoniazid as the reference control (Table ).

9. Tuberculostatic Activity Indices of Epoxybenzo­[7,8]­oxocino­[4,3-b]­pyridine Derivatives 4a–e, 5a–e .

	Minimum concentration of the drug, μg/mL
Compound	100	50	25	12,5	6,25	3,12	1,56	0,78	0,39	0,195	Control
81a	±	+	+	++	+++	+++	+++	+++	+++	+++	+++
81b	±	±	±	+	+	+	+	++	+++	+++	+++
81c	0	±	+	++	++	+++	+++	+++	+++	+++	+++
81d	0	0	0	0	±	±	±	±	±	±	+++
81e	0	0	0	0	0	0	0	±	±	±	+++
82a	+	+	+++	+++	+++	+++	+++	+++	+++	+++	+++
82b	+	++	+++	+++	+++	+++	+++	+++	+++	+++	+++
82c	0	+	++	++	+++	+++	+++	+++	+++	+++	+++
82d	0	0	++	++	+++	+++	+++	+++	+++	+++	+++
82e	++	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++
Isoniazid	0	0	0	0	0	0	0	0	0	0	+++

^a0: Absence of Mycobacterium tuberculosis (MTB) colonies per 100 microscopic fields; ±: 1–9 MTB per 100 microscopic fields; +: 10 to 100 MTB per 100 microscopic fields; ++: 1 to 9 MTB per single microscopic field; +++: 10 or more MTB per single microscopic field.

Based on the results in Table , it was concluded that pyridylchalcones 81d and 81e demonstrated significant tuberculostatic potential by completely inhibiting the growth of M. bovis with MIC values of 1.56 and 12.5, respectively, though they remain less potent than the reference drug, isoniazid.

In, the biological profile of newly synthesized compounds 83a–i was investigated in silico using DFT calculations, PASS Online activity prediction, molecular docking, molecular dynamics simulations, and ADMET profiling via ADMETlab 3.0. DFT-derived molecular electrostatic potential (MEP) mapping of compounds 83a–i revealed the carbonyl oxygen as the predominant nucleophilic site, supported by secondary negative potentials on other O/N heteroatoms, while the pyridine protons constituted the main electrophilic centers (Figure ).

11.

Molecular electrostatic potential (MEP) maps for molecules 83a–i.

To assess the multitarget antiviral potential of Scaffold 2 derivatives 83a–i, their predicted molecular docking-based binding affinities against HRV-14, HIV-1 RT, and SARS-CoV-2 RdRp were calculated and compared with those of benchmark therapeutic agents (Table , Figure ).

10. Binding Affinities of Epoxybenzooxocine Derivatives 83a–i with Viral Proteins: HRV-14 (PDB ID: 4PDW), HIV-1 Reverse Transcriptase (PDB ID: 3V81), and SARS-CoV-2 RdRp (PDB ID: 7AAP) .

	Binding affinity with protein, kcal/mol
Compound	4PDW	3 V81	7AAP
Ribavirin	-6.7	-	-
Nevirapine	-	-10.7	-
Favipiravir	-	-	-6.9
83a	–8.6	–11.5	–9.7
83b	–8.3	–10.2	–9.2
83c	–9.5	–12.4	–10.2
83d	–7.9	–10.4	–8.8
83e	–9.2	–12.1	–9.3
83f	–9.0	–10.8	–10.6
83g	–8.6	–11.3	–9.9
83h	–8.5	–10.5	–9.4
83i	–9.6	–13.4	–10.8

12.

Visualizitaion of protein amino acid–83i ligand interactions.

Docking analysis of compound 83i across the rhinovirus (4PDW), HIV-1 (3V81), and SARS-CoV-2 (7AAP) targets demonstrated dominant H-bonding and π-type interactions, with 7AAP exhibiting the highest degree of stabilization (six H-bonds and two π–cation contacts). The structural flexibility of the central C–C–N linker connecting the epoxybenzooxocinopyridine and methanopyridodiazocine frameworks enables 83i to adopt optimal conformations within various binding sites, complemented by hydrogen bonding from its carbonyl and nitrogen sites and π-stacking from its aromatic rings. Although compound 83i emerged as the most potent candidate in terms of binding affinity and stability toward the HIV-1 protein, a comprehensive ADMET profile revealed significant pharmacological limitations related to its absorption and toxicity. Therefore, compounds 83b, 83d, and 83g were identified as the most promising candidates for further development, offering an excellent balance between predicted antiviral efficacy and favorable metabolic stability.

In summary, the dual-vector functionalization of the epoxybenzooxocinopyridine matrix at the C-3 and C-11 positions provides a clear roadmap for expanding its medicinal chemistry profile. Key structure–activity relationship (SAR) trends demonstrate that targeted modifications at the C-3 positionparticularly quinoxalin-2-one and heterocyclic conjugationsdrastically enhance both antitubercular (M. bovis) and antiviral (SARS-CoV-2) potencies, yielding prominent leads such as compounds 73a, 75, and 81d. Concurrently, C-11 substitution offers an effective pathway to fine-tune multitarget viral binding while improving ADMET characteristics, with candidates 83b, 83d, and 83g displaying an optimal balance between activity and metabolic safety. Collectively, these functionalization strategies establish the epoxybenzooxocine core as a highly versatile pharmacophoric platform for the rational design of next-generation antimicrobial and antiviral agents.

7. Conclusion

This paper provides the first comprehensive review focused on the epoxybenzooxocine core (1) of integrastatins and their analogs, systematically covering their structure, synthetic approaches, functionalization strategies, and bioactivity evaluations reported between 1999 and 2025. Integrastatins A (2a) and B (2b) with an epoxybenzooxocine core were initially isolated from an unnamed fungal source (ATCC 74478) and an Ascochyta species (ATCC 74477) by Singh and coworkers at Merck Research Laboratories in 2002, demonstrating HIV-1 integrase inhibition. Later, in 2013, Laatsch and Proksch isolated two further natural analogs, epicoccolide A (3) and epicocconigrone A (4), which exhibited a broad spectrum of biological activitiesincluding anticancer, antibacterial, and antifungal effectsthereby bringing tetracyclic epoxybenzooxocine pharmacophores back into focus.

From a structural perspective, integrastatins A (2a) and B (2b), along with epicoccolide A (3) and epicocconigrone A (4), are tetracyclic aromatic heterocycles featuring an unprecedented [6/6/6/6] tetracyclic framework, wherein a central [3/3/1] bicyclic ketal core is fused to aromatic rings on both flanks. Due to the inherent rigidity of the central oxocine ring, these molecular scaffolds typically adopt a distinct, nonplanar V-shaped or inverted V-shaped conformation.

According to PubChem molecular descriptors, these natural products comply with Lipinski’s Rule of Five (MW < 500 Da, balanced log P, heavy atom count 27–29), which establishes a baseline filter for drug-likeness, while deeper ADMET profiling further highlights their high predicted human intestinal absorption (HIA) alongside a low risk of cardiotoxicity. Given the severe limitations of extracting these natural compounds from endemic fungal biomass in quantities sufficient for pharmaceutical development, the total synthesis and structural modification of the rare epoxybenzooxocine core became an essential objective. Over the period from 2003 to 2025, researchers globally have driven remarkable progress in this field, successfully advancing from the pioneering but arduous 11-step sequence reported by Foot in 2003 to highly streamlined, atom-economical one-step synthetic protocols developed by Ramana in 2008 and Kulakov in 2021.

A systematic comparative evaluation across key synthetic parameters (including step count, yield, scalability, reactant availability, and scope limitations) reveals that while one-step cascade protocols offer atom economy, their efficiency remains sensitive to electronic and steric constraints imposed by substituents. The decisive role of total synthesis in structural validation was recently demonstrated by Treiber and coworkers (2025), whose total synthesis and NMR analysis corrected a long-standing misinterpretation in the literature, proving that the compound previously assigned as epicoccolide A was in fact epicocconigrone A (4).

Postsynthetic functionalization strategies between 2022 and 2025 yielded a library of more than 30 epoxybenzooxocinopyridine derivatives, enabling a transition toward qualitative structure–activity relationship analysis. Experimental in vitro evaluations established distinct antimicrobial (M. bovis, S. aureus, P. carotovorum, C. albicans), antitubercular, and antiviral (SARS-CoV-2, HIV-1 integrase inhibition) potencies, while computational molecular modeling simulations elucidated binding modes with viral targets including HRV-14, HIV-1 reverse transcriptase, and SARS-CoV-2 RdRp. Integrating pharmacophore mapping with in silico ADMET profiling (Caco-2/MDCK permeability, BBB penetration, PPB, CYP inhibition, and organ toxicity indicators) has defined key vectors for structural optimization, establishing these heterocycles as viable early-stage lead platforms.

In perspective, despite the favorable in silico drug-likeness profiles and synthetic versatility demonstrated across the epoxybenzooxocine framework, several critical bottlenecks must be addressed to translate these structural scaffolds into viable candidates for further drug discovery. First, a fundamental priority is the systematic transition from predictive in silico models (PASS analysis and molecular docking) and primary in vitro screening to comprehensive in vivo pharmacokinetic, bioavailability, and toxicological evaluations to rigorously validate their safety and efficacy profiles. Second, structural biology efforts must focus on target identification and the determination of high-resolution X-ray cocrystal structures of lead derivatives bound to key biological targets, such as HIV-1 and SARS-CoV-2 proteins, thereby transitioning empirical optimization into rationally driven, structure-based drug design. Finally, future synthetic strategy should encompass scaffold hopping through the expansion of heterocyclic fusionsspecifically annelating the core epoxybenzooxocine matrix with diverse nitrogen-containing rings, such as triazoles, pyrazoles, and oxazoles, to systematically broaden both the proprietary patent space and the accessible pharmacological activity landscape.

The authors declare no competing financial interest.

During the preparation of this work, the authors used Grammarly to refine the language of the manuscript. After using this service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.