Chemistry and Antifungal Activity of Homoisoflavonoids

. Tis review deals with the antifungal profle of a subclass of natural products known as homoisofavonoids. Tese molecules belong to the larger favonoids class, yet they are less common for presenting an extra carbon in their basic chemical structures. Homoisofavonoids are important bioactive molecules because they exhibit promising antimutagenic, antiproliferative, anti-oxidant, anti-infammatory, and antimicrobial activity. Tis review lists the principal experimental studies addressing homo-isofavonoid antifungal activity with the aim of discussing the role of these molecules in obtaining new antifungal agents. Te vast majority of research consists of antimicrobial screenings. It was noted that sappanin-type homoisofavonoids commonly exhibit antifungal activity, but their overall antifungal profle is still very little known. Studies evaluating mechanisms of action are needed to better understand the antifungal potential of homoisofavonoids.


Introduction
Homoisofavonoids are a rare and unusual subclass of natural products that form part of the larger family of favonoids. Tey are uniquely characterized by having one more carbon (C-9) in their 16-carbon skeleton than regular favonoids (C 6 C 3 C 6 ) ( Figure 1). Te term homoisofavonoid was initially used to defne the two natural products that were isolated from Eucomis bicolor Bak in 1967, eucomin and eucomol ( Figure 2). Tis denomination was considered inadequate, considering that the compounds are not isofavone derivatives. However, the term homoisofavonoids came to be widely used to defne this group of molecules [1,2].
Te most likely route for the biosynthesis of homoisofavonoids was proposed by Dewick in 1973 and1975, in which it was demonstrated that 2′-methoxychalcones are biosynthetic precursors of the homoisofavonoids. According to Scheme 1, the cyclization of intermediate (II), produced by chalcone (I), would produce either product (III) by the loss of a proton or product (IV) by the addition of a hydride ion. Te experiment of Dewick also indicates that eucomin biosynthesis occurs upon the addition of a carbon atom (derived from methionine) to a chalcone-type skeleton with 15 carbon atoms [3,4].
Each year, fungal infections afect more than a billion people worldwide, with candidiasis representing about 75% to 88% of these infections. Of these, systemic infections are emerging as a major public health problem worldwide. In hospitalized patients, candidemia is the most common form of invasive candidiasis, representing about 9% of all bloodstream infections in the nosocomial environment. Among immunocompromised individuals, the mortality rate from disseminated candidiasis is between 35 and 60%. Te Candida species most commonly involved in invasive candidiasis are C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei [18][19][20][21][22][23][24].
Te major problem lies in the fact that multiresistant strains of non-albicans Candida (C. glabrata, C. krusei, and C. parapsilosis) are increasingly involved in cases of disseminated candidiasis, making the treatment more difcult and consequently increasing mortality rates. Te relatively small number of antifungal agents currently available for therapy, when associated with their indiscriminate use, promotes the development of multidrug-resistant strains, thus leading to the search for new, more potent, and safer antifungal agents [18,25].
Considering the importance of homoisofavonoids, this review discusses their antifungal properties and defnes (through an evaluation of scientifc articles published between 2002 and 2022) their place in the search for new molecules with antifungal profles.

Methodology
Tis literature review was performed using the PubMed and Scopus databases. Te search period selected was from January 2002 to August 2022. Te following keywords were used: homoisofavonoids, antifungal activity, antifungal agents, antimicrobial activity, and antimicrobial agents. Scientifc articles concerning experimental research published in English were selected. An initial analysis of the title was performed followed by the evaluation of the abstract and using as a selection criterion: performance of antifungal and/ or antimicrobial tests with natural or synthetic homoisofavonoids.

Results and Discussion
Few studies addressing the antifungal activity of natural or synthetic homoisofavonoids have been published over the last 20 years (2002-2022). Table 1 summarizes the principal scientifc articles selected by this review to discuss the antimicrobial activity and especially the antifungal capacity of homoisofavonoids. In general, the studies used in vitro assays involving microdilutions in broth with a defnition of the minimum inhibitory concentration (MIC) or the agar cup bioassay with the delimitation of homoisofavonoid fungal growth inhibition zones. Species of the genus Candida, mainly C. albicans, were widely used in the antifungal tests, and considering the increasing number of serious fungal infections involving Candida species, these data are indeed relevant. Te vast majority of homoisofavonoids discussed were isolated from difering species of the genus Caesalpinia, which in the diferent regions where they were cultivated enjoyed popular use in treating various health problems, including superfcial and mucosal infections.
In 2008, Rivero-Cruz presented relevant results for the antimicrobial activity of the homoisofavonoids (Figure 4), hematoxylin (6) and brazilin (3), against C. albicans Ca 54 and various bacterial species involved in human infections. Te compounds were isolated from Haematoxylon brazilian Karst. (Leguminosae), a large tree, abundant in southeastern Mexico. Tea from the bark of the plant is popularly used to treat stomach discomfort, hypertension, and oral infections, among other conditions. In vitro antimicrobial assays were performed for the derivatives using the agar difusion method, and minimum inhibitory concentrations (MIC) were measured using the broth microdilution method. Te antibacterial tests used strains of methicillin-resistant Staphylococcus aureus, Escherichia coli, and Enterococci faecium. Te results revealed that the homoisofavonoids were capable of inhibiting microorganism growth, including C. albicans at a threshold concentration of >128 μg/mL. Compounds 3 and 5 were less potent than the other isolated compounds (gallic acid, methyl gallate, and 4-hydroxycinnamic acid); however, they presented a broad antimicrobial spectrum [10].
Das et al. obtained homoisofavonoids by extraction and isolation from the aerial parts of Caesalpinia pulcherrima (a small leguminous tree found throughout India). Te authors prepared homoisofavonoid derivatives through condensation reactions between 7-hydroxy-4-chromanone or 7methoxy-4-chromanone and difering substituted aldehydes, a process promoted using a piperidine base (Scheme 2). All of the derivatives were evaluated for antimicrobial activity using an agar cup bioassay methodology with measurement of microorganism growth inhibition zones after 48 h. Te derivatives presented bioactivity against Journal of Chemistry   from southern Africa traditionally used for infection, infammation, pain/rheumatic fever, and as a laxative, among others). Te fungal species used in the study to defne the minimum inhibitory concentration was Saccharomyces cerevisiae. However, the homoisofavonoids did not exhibit antifungal activity. Alali et al. also evaluated sappanin-type homoisofavonoids (isolated from the bulb of Bellevalia eigii Feinbrun) against strains of C. albicans and Aspergillus niger without promising results [30,31]. Te most recent antifungal activity results for homoisofavonoid derivatives are from an experimental study in our research group, in which Ferreira et al. observed moderate antifungal bioactivity for synthetic homoisofavonoids (sappanin-type) against Candida species. Te tested homoisofavonoids were prepared using an aldol condensation process between 4-chromanone and difering substituted aldehydes, with pyrrolidine as a catalyst, as shown in Scheme 3 [28].
In the microdilution test (CLSI, 2008), derivative 19 presented moderate bioactivity, inhibiting the growth of C. albicans (ATCC 90028), C. tropicalis (ATCC 13803), and C. krusei (ATCC 6258) strains at the concentration of 62.5 μg/ml. Compound 19 also exhibited fungicidal capacity against these strains at the same concentration. Te study suggests that its bioactivity was infuenced by the substituent m-OCH 3 on aromatic ring B [31].
Trough in vitro tests using the broth microdilution method, Ferreira et al. as mentioned above demonstrated that the antifungal action of homoisofavonoid 17 does not occur through direct interaction with components of the C. albicans fungal plasma membrane or with the cell wall. More advanced studies are needed to clarify the mechanism of action and the relationship between homoisofavonoid 17′s chemical structure and its antifungal profle [31].

Conclusions
In the present review, reports of homoisofavonoid antifungal activity against various types of fungi were discussed. Te small number of reports displays the lack of either phytochemical or synthetic studies involving this chemical class. However, it is important to highlight that in the studies addressed in this review, homoisofavonoids of the sappanin-type, which are the most common in nature, do exhibit antifungal activity. Tis indicates that more research studies in this feld are needed to better understand which structural characteristics of the basic homoisofavonoid skeleton are important and/or essential for their antifungal properties and which molecular modifcations might be carried out to optimize this bioactivity. Such bioactive compounds can be used as molecular models or prototypes for the design and synthesis of more potent structural analogues. Investigation of mechanisms of action and standardization of experimental protocols is essential to reveal the potential of these compounds as antifungal drug candidates.

Data Availability
Te data supporting this review are from previously reported studies and datasets, which have been cited.  Journal of Chemistry