Guanidine-Containing Polyhydroxyl Macrolides: Chemistry, Biology, and Structure-Activity Relationship.

Antimicrobial resistance has been seriously threatening human health, and discovering new antimicrobial agents from the natural resource is still an important pathway among various strategies to prevent resistance. Guanidine-containing polyhydroxyl macrolides, containing a polyhydroxyl lactone ring and a guanidyl side chain, can be produced by many actinomycetes and have been proved to possess many bioactivities, especially broad-spectrum antibacterial and antifungal activities. To explore the potential of these compounds to be developed into new antimicrobial agents, a review on their structural diversities, spectroscopic characterizations, bioactivities, acute toxicities, antimicrobial mechanisms, and the structure-activity relationship was first performed based on the summaries and analyses of related publications from 1959 to 2019. A total of 63 guanidine-containing polyhydroxyl macrolides were reported, including 46 prototype compounds isolated from 33 marine and terrestrial actinomycetes and 17 structural derivatives. Combining with their antimicrobial mechanisms, structure-activity relationship analyses indicated that the terminal guanidine group and lactone ring of these compounds are vital for their antibacterial and antifungal activities. Further, based on their bioactivities and toxicity analyses, the discovery of guanidyl side-chain targeting to lipoteichoic acid of Staphylococcus aureus indicated that these compounds have a great potency to be developed into antimicrobial and anti-inflammatory drugs.

1. Introduction

Antimicrobial resistance has become a serious threat to human health and economic development [1]. Many strategies involving the development of new antimicrobial agents [2], the revival of old antibiotics [3,4], and combination therapy had been putting forward to fight or delay resistance [5]. On the one hand, our group has been researching the practice and law of drug combinations to prevent antimicrobial resistance [5,6,7]; on the other, we have been trying our best to discover new antimicrobial agents. Guanidine-containing polyhydroxyl macrolides can be generally biosynthesized by many actinomycetes [8,9,10], and all these compounds contain a lactone ring and a guanidyl side chain. Azalomycin F, a complex including three main compounds, F3a, F4a, and F5a, isolated from the broth of Streptomyces hygroscopicus var. azalomyceticus [11,12,13,14], was the first one reported. The planar structures of these three compounds were established by Namikoshi, Iwasaki, and Chandra et al. from 1982 to 1995 [13,14,15,16,17], and revised by Yuan et al. in 2011 [18]. Contemporaneously, many other guanidine-containing polyhydroxyl macrolides [19,20,21,22,23], such as niphimycin, RS-22, guanidylfungins, amycins, and shurimycins, were isolated from actinomycetes, especially streptomycetes. Although the planar structures of these compounds have been elucidated, their stereochemistries remained undetermined except the E-configuration of double bonds. Until 2013, the relative configurations of azalomycins F5a, F4a, and F3a as three representatives of these compounds, together with seven new analogs, were reported by Yuan et al. [24,25]. Moreover, antimicrobial mechanisms indicated that the cell membrane was the main action site of these compounds against bacteria and fungi and that they could change the plasma membrane permeability and lead to the leakage of cellular substances [26,27,28,29]. In 2019, Yuan et al. [30] reported that azalomycin F5a could bind to the polar head of cell-membrane phospholipid and target to lipoteichoic acid (LTA) to kill methicillin-resistant Staphylococcus aureus (MRSA). Up to 2019, approximately 48 guanidine-containing polyhydroxyl macrolides have been isolated from 33 marine and terrestrial actinomycetes. Moreover, 15 structural derivatives were synthesized. These compounds have broad-spectrum antimicrobial activity (especially Gram-positive bacteria and fungi) [31,32,33], anti-trichomonas [34], cytotoxicity [31,32], and so on. The structures and antimicrobial activities of some of them were partly involved in two previous reviews [31,32], while the complete structural diversity and bioactivities, acute toxicities, antimicrobial mechanisms, and structure-activity relationship of these compounds were not yet reported. Along with the clarification of some antimicrobial mechanisms and structure-activity relationships, we here presented a review on the chemistry and biology of these compounds discovered from 1959 to 2019 for exploring their potential in drug development.

2. Structural Diversity

Guanidine-containing polyhydroxyl macrolides are widely produced by actinomycetes, especially streptomycetes. Depending on the atom number composed of the lactone ring, they can be classified into three types: 32-, 36- and 40-membered polyhydroxyl macrolides. Until 2019, 48 guanidine-containing polyhydroxyl macrolides have been isolated and identified, which include seven 32-membered (1–7, Figure 1), thirty-six 36-membered (8–43, Figure 2), and five 40-membered (44–48) macrolides (Figure 3). All these compounds have a guanidyl side chain and a lactone ring, which includes a six-membered hemiketal ring, while there are differences at (1) the chain length (nine or eleven carbons), methyl number (one or two), and double bond position of guanidyl side chains [35]; (2) the atom number composed of the lactone ring (32, 36, or 40); (3) the number of malonyl monoesters (1 or 2), and their sites (C-19, C-23, or C-25) linking at the lactone ring; (4) the hydroxyl, methyl, and double bond numbers of the lactone ring. Simultaneously, all hydroxyl and methyl groups, except for those on six-membered hemiketal ring, present 1,3-, 1,5-, 1,7-, or 1,9- substitution characteristics, which include 1,3,5-, 1,5,7-, 1,3,5,7-, and 1,3,5,7,9- substitutions, and so on, and double bonds or carbonyl groups can be considered as a potential hydroxyl group as they can be, respectively, formed by the dehydration or oxidation of hydroxyl groups. These structural characterizations can be interpreted by the biosynthesis of guanidine-containing macrocyclic polyketides [8,9,10] and are very helpful for the structural elucidation of these compounds. However, the natural existence of compounds 37 and 39 cannot be explained as they share the same origin for guanidinobutanoate starter units in their biosynthesis pathways [9].

Figure 1

Chemical structures of 32-membered guanidine-containing polyhydroxyl macrolides (1–7).

Figure 2

Chemical structures of 36-membered guanidine-containing polyhydroxyl macrolides (8–43).

Figure 3

Chemical structures of 40-membered guanidine-containing polyhydroxyl macrolides (44–48).

Moreover, compound RP 63834 (48, Figure 3) has a 41-membered lactone ring and a guanidyl side-chain containing eight carbons [36], which is different from the general character (a 32-, 36-, or 40-membered ring and a 9- or 11-carbon guanidyl side-chain) of other polyhydroxyl macrolides and is not in accordance with the rule that the positions of the ketone group are (n−2)/2 position in n-membered macrocyclic lactones [35]. Maybe, one of three methylene at C46, C48, and C49 position [36] should be assigned to its guanidyl side-chain, and compound RP 63834 is a 40-membered polyhydroxyl macrolide. Thereby, we deduced that compound RP 63834 was likely a compound 44. The numbers, names, and corresponding sources and references of all these compounds are shown in Table 1.

Table 1

Guanidine-containing polyhydroxyl macrolides from a natural source.

Compounds	Name	Sources	References(Publication Time)
1	Copiamycin A	Streptomyces hygroscopicus var. crystallogenes ATCC 19040	[47] (1965)
		Streptomyces violaceoniger TÜ 905	[39] (1981)
		Streptomyces hygroscopicus sp. M931-1	[48] (1991)
		Actinomycete MT2617-2	[40] (1996)
		Streptomyces hygroscopicus var. crystallogenes IFM 1136	[49] (1999)
2	Neocopiamycin A	Streptomyces hygroscopicus var. crystallogenes	[50] (1984)
		Streptomyces hygroscopicus var. crystallogenes IFM 1136	[49] (1999)
3	Neocopiamycin B	Streptomyces hygroscopicus var. crystallogenes IFM 1136	[49] (1999)
4	Demalonylcopiamycin	Streptomyces hygroscopicus var. crystallogenes IFM 1136	[49] (1999)
5	Demalonylmethylcopiamycin	Streptomyces hygroscopicus var. crystallogenes IFM 1136	[49] (1999)
6	Guanidolide A	Streptomyces hygroscopicus var. crystallogenes IFM 1136 Streptomyces	[49] (1999)
		hygroscopicus var. crystallogenes	[51] (1988)
7	TMC-34	Streptomyces. A-3030	[52] (1995)
8	Azalomycin F3a	Streptomyces hygroscopicus var. azalomyceticus	[11] (1960)
		Streptomyces hygroscopicus MSU/MN-4-75B	[17] (1995)
		Streptomyces malaysiensis MJM1968	[36] (2010)
		Streptomyces sp. 211726	[18] (2011)
9	Azalomycin F4a	Streptomyces hygroscopicus var. azalomyceticus,	[11] (1960)
		Streptomyces hygroscopicus MSU/MN-4-75B	[17] (1995)
		Streptomyces malaysiensis MJM1968,	[36] (2010)
		Streptomyces sp. 211726	[18] (2011)
10	Azalomycin F5a	Streptomyces hygroscopicus var. azalomyceticus,	[11] (1960)
		Streptomyces hygroscopicus MSU/MN-4-75B	[17] (1995)
		Streptomyces malaysiensis MJM1968,	[36] (2010)
		Streptomyces sp. 211726	[18] (2011)
11	2-Demethyl azalomycin F4a	Actinomycete sp. HIL Y-9120362	[39] (1995)
12	2-Demethyl azalomycin F5a	Actinomycete sp. HIL Y-9120362	[39] (1995)
13	25-Malonyl demalonyl azalomycin F5a monoester	Streptomyces sp. 211726,	[25] (2013)
14	23-Valine demalonyl azalomycin F5a ester	Streptomyces sp. 211726,	[25] (2013)
15	23-(6-Methyl) heptanoic acid demalonylazalomycin F3a ester	Streptomyces sp. 211726,	[25] (2013)
16	23-(6-Methyl) heptanoic acid demalonylazalomycin F4a ester	Streptomyces sp. 211726,	[25] (2013)
17	23-(6-Methyl) heptanoic acid demalonylazalomycin F5a ester	Streptomyces sp. 211726,	[25] (2013)
18	23-(9-Methyl) decanoic acid demalonylazalomycin F4a ester	Streptomyces sp. 211726,	[25] (2013)
19	23-(10-Methyl) undecanoic acid demalonylazalomycin F4a ester	Streptomyces sp. 211726,	[25] (2013)
20	RS-22A	Streptomyces violaceusniger RS-22	[22,38] (1995)
21	RS-22B	Streptomyces violaceusniger RS-22	[22,38] (1995)
22	RS-22C	Streptomyces violaceusniger RS-22	[22,38] (1995)
23	Azalomycin F4a 2-ethylpentyl ester	Streptomyces sp.211726	[18] (2011)
24	Azalomycin F5a 2-ethylpentyl ester	Streptomyces sp.211726	[18] (2011)
25	Shurimycin A	Streptomyces hygroscopicus A1491	[23] (1992)
26	Shurimycin B	Streptomyces hygroscopicus A1491	[23] (1992)
27	Amycin B	Streptomyces sp. DSM 3816,	[21] (1990)
		Streptomyces sp. IMB7-145	[53] (2018)
28	Niphimycin (scopafungin)	Streptomyces hygroscopicus B-255	[54] (1967)
		Streptomyces sp. DSM 3816,	[21] (1990)
		Streptomyces sp.KP6107,	[28] (2013)
		Streptomyces sp. GE48009	[55] (1997)
		Streptomyces hygroscopicus var. enhygrus	[56,57] (1972)
		Streptomyces hygroscopicus var. enhygrus NRRL 3664	[58] (1986)
		Streptomyces hygroscopicus var. enhygrus var. nova UC-2397	[59] (1971)
		Streptomyces sp. IMB7-145	[53] (2018)
29	Amycin A	Streptomyces sp. DSM 3816	[21] (1990)[53] (2018)
30	25-Malonyl Demalonylniphimycin	Streptomyces sp. IMB7-145	[53] (2018)
31	19,25-Malony Demalonylniphimycin	Streptomyces sp. IMB7-145	[53] (2018)
32	15-Malonyl Niphimycin	Streptomyces sp. IMB7-145	[53] (2018)
33	17-O-Methylniphimycin	Streptomyces sp. IMB7-145	[53] (2018)
34	N′-methyniphimycin	Streptomyces spec. 57-13	[60] (1998)
35	Guanidyfungina A	Streptomyces hygroscopicus No. 662	[20,61] (1984)
36	Guanidyfungina B	Streptomyces hygroscopicus No. 662	[20,61] (1984)
37	Kanchanamycin A	Streptomyces strain Tü 4018,Streptomyces lasiicapitis sp. nov.	[38,62] (1996)[35] (2017)
38	Kanchanamycin C	Streptomyces strain Tü 4018,Streptomyces lasiicapitis sp. nov.	[38,62] (1996)[35] (2017)
39	Kanchanamycin D	Streptomyces strain Tü 4018,Streptomyces lasiicapitis sp. nov.	[38,62] (1996)[35] (2017)
40	Malonyl-4,5-dihydroniphimycin	Streptomyces hygroscopicus 15	[63] (2007)
41	Dihydroniphimycin	Streptomyces hygroscopicus 15	[64] (2000)
42	Polaramycin A	Streptomyces hygroscopicus LP-93	[65] (1997)
43	Polaramycin B	Streptomyces hygroscopicus LP-93	[65] (1997)
44	Malolactomycin A	Streptomyces sp. 83-634	[45,66] (1993)
45	Malolactomycin B	Streptomyces sp. 83-634	[45] (1993)
46	Malolactomycin C	Streptomyces KP-3144	[67] (1997)
47	Malolactomycin D	Streptomyces KP-3144	[67] (1997)
48	RP 63834	Streptomyces strain n* S-13361	[36] (1991)

Among 33 guanidine-containing polyhydroxyl macrolide-producing strains (Table 1), sixteen belong to Streptomyces hygroscopicus, ten are unidentified species of streptomycete genus, and other strains are Streptomyces lasiicapitis 3H-HV17(2)T [37], Streptomyces malaysiensis MJM1968 [27], Streptomyces olivaceus Tü 4018 [38], Streptomyces violaceoniger TÜ 905 [39], Streptomyces violaceusniger RS-22 [40], and two actinomycete strains HIL Y-9120362 and MT2617-2 [41,42]. To understand the affinity of these strains, the phylogenetic tree (Figure 4) was constructed using the neighbor-joining algorithms (some similar strains belonging to the same species of these strains, which have no 16S rRNA gene sequences, were used) [43]. Briefly, their 16S rRNA gene sequences were aligned against sequences of reference strains using the BLAST program (http://www.ncbi.nlm.nih.gov/). All the selected DNA multiple sequences were matched by means of software package Clustal_X 1.83 [44], and evolution distances were calculated using the Kimura2-Parameter model of MEGA version 6.0 [45]. Based on 1000 replicates, the confidence coefficient of the phylogenetic tree was evaluated using bootstrap analysis [46]. From Figure 4, there are no obvious distribution rules of species and genera between these guanidine-containing polyhydroxyl macrolides and their producing strains. Namely, the same macrolide can be produced by several species and genera with different genetic distances.

Figure 4

Neighbor-joining tree based on the 16S rRNA gene sequences of some strains producing guanidine-containing polyhydroxyl macrolide. Some similar strains belonging to the same species of these strains, which have no 16S rRNA gene sequences, were used.

These molecules contain many chair centers (more than eighteen). As they have lager flexibility attributed to the larger ring and the longer side-chain, their single crystals were hardly obtained for determining the stereochemistry using an x-ray single diffraction method [15]. Simultaneously, other methods [68], such as optical rotatory dispersion (ORD), vibrational circular dichroism (VCD), and electrostatic circular dichroism (ECD), were also difficult to have assigned their absolute configurations because of the complexity and flexibility of these compounds. Thereby, most compounds only presented their planar structures except for the relative configurations of azalomycin F analogs and derivatives (8–10, 13–19) [24,25] and the proposed absolute configurations of niphimycin analogs (27–33) [53].

Moreover, 15 derivatives (49–63, Figure 5) were synthesized from copiamycin, azalomycin F, guanidylfungin A, and niphimycin, which mainly involved the etherification of hemiketal hydroxyl and/or the hydrolysis of malonyl moiety. Their numbers, names, and corresponding raw materials and references are listed in Table 2.

Figure 5

Structural derivatives (49–63) of some guanidine-containing polyhydroxyl macrolides.

Table 2

Structural derivatives (49–63) of some guanidine-containing polyhydroxyl macrolides.

Compounds	Derivatives Name	Raw Materials	References(Publication Time)
49	17-Methyl copiamycin	Copiamycin	[69] (1985)
50	17,29-Dimethyl demalonylazalomycin F4a	Azalomycin F4a	[58] (1986)
51	17-Methyl demalonylazalomycin F5a	Azalomycin F5a	[70] (2014)
52	17-Ethyl demalonylazalomycin F5a	Azalomycin F5a	[70] (2014)
53	17-Butyl demalonylazalomycin F5a	Azalomycin F5a	[70] (2014)
54	17-Allyl demalonylazalomycin F5a	Azalomycin F5a	[70] (2014)
55	Demalonylazalomycin F3a	Azalomycin F3a	[71] (2016)
56	Demalonylazalomycin F4a	Azalomycin F4a	[71] (2016)
57	Demalonylazalomycin F5a	Azalomycin F5a	[71] (2016)
58	17-Methyl guanidylfungin A	Guanidylfungin A	[69] (1985)
59	17-Ethyl guanidylfungin A	Guanidylfungin A	[69] (1985)
60	17-Butyl guanidylfungin A	Guanidylfungin A	[69] (1985)
61	17-Allyl guanidylfungin A	Guanidylfungin A	[69] (1985)
62	17-Methyl demalonylguanidylfungin A	Guanidylfungin A	[69] (1985)
63	17-Methyl demalonylniphimycin	Niphimycin	[58] (1986)

3. Spectroscopic Characterization

As the chemical structures of these compounds have many similar fragments and groups, many NMR signals are close to each other, and some even overlap. These will increase the difficulties of their structural elucidations, while some regularity spectroscopic characterizations could be summarized, which would be very conducive to the structural elucidations of these compounds.

For the guanidyl side-chain, their guanidyl carbon signals at 157.3 to 158.3 ppm are easily observed from their 13C NMR spectra, and their chemical shifts decrease from approximately 158.7, 158.3 to 157.3 when the methyl number linking with guanidino nitrogen increases from 0, 1 to 2 [18,22,25,53]. These are also confirmed by the proton signals of N-methyl at 2.8 to 2.9 ppm on the 1H NMR spectra. Moreover, the stereochemistry of chain double bond is generally oriented in E-configuration. This is hardly established from the NMR spectrometric data due to overlapping signals; however, it can be confirmed by comparing the band at 969 cm−1 in the IR spectrum with the spectral data in the book [18,72].

For the lactone ring, many methyl and oxygenated methine signals can be observed from their 13C NMR spectra. As all these compounds share polyketide biosynthesis pathway [8,9,10,53], some general substitution characteristics of the hydroxyls and methyl groups linking on the lactone ring and guanidyl side-chain, such as 1,3-, 1,5-, 1,7-, 1,9-, 1,3,5-, 1,3,5,7-, 1,5,7-, and 1,3,5,7,9- substitutions, are very useful for their structural elucidations and NMR signal assignments. Some NMR data, including a quaternary hemiketal carbon at 99 to 100 ppm, the carbon at about 80 ppm and proton at 4.81 ppm of oxygenated methine forming lactone, and the proton at 5.23 ppm of oxygenated methine linking malonyl moiety will be also helpful for their structural elucidations. Furthermore, the presence or not of a conjugated diene and/or an α, β, γ, and δ-unsaturated acid (or ester) group can be easily deduced from whether there are UV absorption maxima at 240 nm (lgε more than 4) and/or 269 nm (lgε more than 4) [13,18]. For the malonyl moiety, the carbon signal of methylene is hardly observed (sometimes a little) in a protic solvent, such as methanol-d4, as the keto-enol tautomerization rapidly occurs [18,53], while it is easily detected in an aprotic solvent, such as DMSO-d6. Simultaneously, the two protons present multiple peaks in the 1H NMR spectrum as they quickly exchange with deuterium at the measurement conditions, especially at higher temperatures [18].

4. Bioactivity

Guanidine-containing polyhydroxyl macrolides have been experimentally documented to possess broad-spectrum antibacterial and antifungal activities, and they can remarkably inhibit the growth of Gram-positive bacteria, yeast, and fungi (Table 3) [25,28,34,38,51,53,58,64]. Moreover, they also show antitrichomonal and antitumor activities [25,73,74].

Table 3

Antibacterial activity of guanidyl-polyol macrolide antibiotics.

Compounds	Minimum Inhibitory Concentrations to Various Pathogenic Microorganisms (μg/mL)	References
1	Sarcina flava IFM 2242 (25), Sarcina flava IFM 2243 (3.12), 3 isolates of Sarcina hansenii (12.5–25.0), 4 isolates of Sarcina lutea (3.12–12.5), Sarcina subflava IFM 2116 (3.12), 3 isolates of Sarcina ureae (1.56–6.25), 16 isolates of Candida albicans (0.78–100), 3 isolates of Candida guilliermondii (100), Candida tropicalis IFM 40018 (100), Candida krusei IFM 40019 (100), Candida parapsilosis IFM 40020 (100), Candida stellatoidea IFM 40021 (100), Cryptococcus neoformans IFM 40037 (1.56), C. neoformans IFM 40038 (1.56), C. neoformans IFM 40047 (6.25), Staphylococcus aureus FDA 209P (12.5), Geotrichum candidum IFM 40068 (1.56), Epidermophyton floccosum IFM 40747 (0.39), Microsporum canis (3.13), Microsporum gypseum IFM 40727 (6.25), Mucor hiemalis (3.13), Mucor Racemosus (3.13), Trichophyton mentagrophytes Kamiyama (3.13), Trichophyton rubrum IFM 40732 (3.13), Sporothrix schenckill IFM 40750 (hyphal phase) (50), Fonsecaea pedrosoi IFM 40756 (100), Histoplasnma capsulatum IFM 40752 (hyphal phase) (0.78), 4 clinical isolates of Trichophyton glabrata (0.78~3.13), 14 clinical isolates of Trichophyton vaginalis (12.5 to >100)	[39,50,51,69,76,77]
2	8 isolates of C. albicans (1.56–6.25), C. guilliermondii IFM 40017 (3.13), C. tropicalis IFM 40018 (3.13), C. krusei IFM 40019 (3.13), C. parapsilosis IFM 40020 (3.13), C. tropicalis (3.13), C. stellatoidea (IFM 40021) (3.13), Candida utillis IFM 40099 (12.5), C. neoformans IFM 40037 (<0.78), C. neoformans IFM 40038 (<0.78), C. neoformans IFM 40047 (1.56), G. candidum IFM 40068 (1.56), Torulopsis glabrata IFM 40065 (6.25), Trichophyton cutaneum IFM 40066 (6.25), Saccharomyces cerevisiae sake IFM 40025 (12.5), S. schenckill IFM 40751 (yeast phase) (3.13), Aspergillus flavus 23 (6.25), Aspergillus fumigatus 25 (25), Aspergillus nidulans 21 (6.25), Aspergillus niger 22 (6.25), Aspergillus oryzae IFM 40607 (6.25), Aspergillus versicolor 26 (12.5), Penicillium expansum IFM 40619 (3.13), E. floccosum IFM 40747 (0.78), M. canis (1.56), M. gypseum IFM 40727 (1.56), T. mentagrophytes IFM 40737 (1.56), T. mentagrophytes Kamiyama (1.56), T. rubrum IFM 40732 (0.39), S. schenckill IFM 40750 (hyphal phase) (6.25), F. pedrosoi IFM 40756 (3.13), H. capsulatum IFM 40752 (hyphal phase) (0.78)	[50]
3	A. flavus 23 (6.25), A. fumigatus 25 (12.5), A. nidulans 21 (6.25), A. niger 22 (6.25), A. oryzae IFM-40607 (6.25), A. versicolor 26 (6.25), Penicillium glaucum 3-1 (12.5), E. floccosum IFM 40747 (<0.78), M. canis (3.12), M. gypseum IFM 40727 (3.12), T. mentagrophytes IFM 40737 (1.56), T. rubrum IFM 40732 (1.56), Sporothrix schenckii IFM 40750 (6.25), S. schenckii IFM 40751 (yeast phase) (6.25), F. pedrosoi IFM 40756 (3.12), 8 isolates of C. albicans (0.78~12.5), C. guilliermondii IFM 40017 (6.25), C. tropicalis IFM 40018 (6.25), C. krusei IFM 40019 (6.25), Candida prarsilosis IFM 40020 (6.25), C. stellatoidea IFM 40021 (3.12), C. neoformans IFM 40037 (<0.78), C. neoformans IFM 40038 (<0.78), C. neoformans IFM 40047 (<0.78), C. utillis IFM 40099 (6.25), T. glabrata IFM 40065 (<0.78)	[49]
4	S. aureus FDA 209P (12.5), Bacillus subtillis NIHJ PCI 219 (12.5), Cochiobolus miyabeanus IFO 5277 (3.2), Alternaria mali IFO 8984 (12.5), Botryotinia fuckeliana IFO 5363 (1.6), Colletorichum lagenarium IFO 7513 (1.6), Pellicularia filamentosa sp. Sasakill IFO 6258 (1.6), Pyricularia oryzae IFO 5994 (6.25), A. oryzae IFO 5239 (25), T. mentagrophytes IFO 6202 (6.25), T. glabrata IFM 40065 (<0.78), S. cerevisiae IFO 0304 (1.6), C. albicans IFO 1594 (1.6), C. krusei IFM 40019 (6.25), C. prarsilosis IFM 40020 (6.25), C. stellatoidea IFM 40021 (3.12), C. neoformans IFM 40037 (<0.78), C. neoformans IFM 40038 (<0.78), C. neoformans IFM 40047 (<0.78), C. utillis IFM 40099 (6.25)	[51]
5	S. aureus FDA 209P (3.12), S. cerevisiae IAM 4020 (6.25), C. albicans, A. nidulans 21 (12.5), P. expansum IFM 40619 (6.25), T. mentagrophytes IFM 40734 (0.78), M. gypseum IFM 40727 (0.78), E. floccosum IFM 40747 (0.78)	[51,69]
6	A. nidulans 21 (50), Paecilomyces expansum (100), T. mentagrophytes IFM 40734 (12.5), M. gypseum IFM 40727 (25), E. floccosum IFM 40747 (12.5)	[51]
7	C. albicans ATCC 48130 (3.1), C. neoformans 145 A (1.6), A. fumigatus TUKUBA 48130 (3.1), T. mentagrophytes (3.1), T. rubrum (1.6)	[52]
8	Ten isolates of S. aureus (4–8), B. subtilis PCI 219 (12.5), S. aureus 209 P (6.25–12.5), S. lutea (6.25), Corynebacterium xerosis (6.25), Mycobacterium smegmatis ATCC607 (25), C. albicans Yu 1200 (1.56), S. cerevisiae (1.56–3.12), Torula utilis (1.56–3.12), C. neoformans (1.56), Kloeckera africana (1.56), Trichophyton asteroids (3.12), Trichophyton interdigitale (1.56), A. oryzae (6.25), A. niger (6.25~12.5), Penicillium notatum (1.56–3.12), P. oryzae (0.78–1.56), Ophioborus miyabeanus (0.78), Alternaria kikuchiana (1.56), Sclerotinia libertiana (0.78–1.56), Fusarium lycopersici (3.12), Fusarium lini (1.56~3.12), Ceratostomella fimbriata (1.56), Trichomonas vaginalis (12.5)	[12,78]
9	Ten isolates of S. aureus (4~12.5), C. albicans (12.5), A. fumigatus IAM 2046 (25), A. fumigatus IAM 2046 (MCC, >200)	[58,78]
10	Ten isolates of S. aureus ATCC 33592 (4~8)	[78]
11	The diameter of the zone of inhibition (mm): C. albicans (11), Penicillium digitatum (15), Fusarium culmorum 100 (17), A. mali P37 (20), P. oryzae K02 (16), Leptosphaeria oryzae J02 (23), Pellicularia sasakill J03 (28), Pseudomonas herpotrichoides 008 (19), Neurospora crassa SGF-18 (11), Botrytis cinereal E02 (22), B. cinereal A06 (13), B. cinereal D01 (17), Phytophthora infestans J08 (13)	[41]
12	The diameter of the zone of inhibition (mm): C. albicans (slight), P. digitatum (14), F. culmorum 100 (16), A. mali P37 (18), P. oryzae K02 (20), L. oryzae J02 (27), P. sasakill J03 (25), P. herpotrichoides 008 (16), N. crassa SGF-18 (12), B. cinereal E02 (20), B. cinereal A06 (12), B. cinereal (D01) (13), P. infestans J08 (15)	[41]
13	C. albicans ATCC 10231 (3.13), S. aureus S014 (0.39), B. subtilis S028 (0.20), Esherichia coli S002 (3.13)	[25]
14	C. albicans ATCC 10231 (6.25), S. aureus S014 (1.56), B. subtilis S028 (0.39), E. coli S002 (6.25)	[25]
15	C. albicans ATCC 10231 (3.13), S. aureus S014 (0.78), B. subtilis S028 (0.39), E. coli S002 (3.13)	[25]
16	B. subtilis S028 (0.20), C. albicans ATCC 10231 (1.56), E. coli S002 (6.25), S. aureus S014 (1.56)	[25]
17	B. subtilis S028 (0.78), C. albicans ATCC 10231 (1.56), E. coli S002 (12.5), S. aureus S014 (0.78)	[25]
18	B. subtilis S028 (0.39), C. albicans ATCC 10231 (3.13), E. coli S002 (25), S. aureus S014 (0.39)	[25]
19	B. subtilis S028 (0.39), C. albicans ATCC 10231 (3.13), E. coli S002 (3.13), S. aureus S014 (0.39)	[25]
20	S. aureus (6.25~12.5), Streptococcus pyogenes Cook (25), C. neoformans KC-201 (3.13), C. albicans KC-07 (3.13), C. tropicalis KC-104 (1.56), C. prarsilosis KC-110 (6.25), C. glabrata KC-308 (6.25), A. flavus KA-06 (12.5), A. fumigatus KA-01 (12.5), T. mentagrophytes KD-114 (12.5), T. rubrum KD-114 (12.5), M. canis KD-305 (12.5), M. gypseum KD-318 (12.5)	[40]
21	S. aureus (6.25~12.5), S. pyogenes Cook (12.5), C. neoformans KC-201 (3.13), C. albicans KC-07 (3.13), C. tropicalis KC-104 (3.13), C. prarsilosis KC-110 (6.25), C. glabrata KC-308) (6.25), A. flavus KA-06 (12.5), A. fumigatus KA-01 (12.5), T. mentagrophytes KD-114 (12.5), T. rubrum KD-114 (12.5), M. canis KD-305 (12.5), M. gypseum KD-318 (12.5)	[40]
22	S. aureus (6.25~12.5), S. pyogenes Cook (25), C. neoformans KC-201 (3.13), C. albicans KC-07 (6.25), C. tropicalis KC-104 (3.13), C. prarsilosis KC-110 (6.25), C. glabrata KC-308 (6.25), A. flavus KA-06 (12.5), A. fumigatus KA-01 (25), T. mentagrophytes KD-114 (12.5), T. rubrum KD-114 (12.5), M. canis KD-305 (12.5), M. gypseum KD-318 (12.5)	[40]
23	C. albicans ATCC 10231 (2.34)	[18]
24	C. albicans ATCC 10231 (12.5)	[32]
25	B. subtillis (3.1), S. lutea (3.1), S. aureus 209P (1.56), Botrytis fragilis (6.2), C. neoformans (1.56), T. mentagrophytes (3.1), A. fumigatus (3.1), A. mali (3.1), Fusarium oxysporum (12.5), B. cinereal (0.78), P. oryzae (0.78), Rhizoctonia solani (0.78), C. albicans (3.1)	[23]
26	B. subtillis (3.1), S. lutea (3.1), S. aureus 209P (1.56), Bacteroides fragilis (12.5), C. neoformans (1.56), T. mentagrophytes (3.1), A. fumigatus (6.2), A. mali (3.1), F. oxysporum (12.5), B. cinereal (0.78), P. oryzae (0.78), R. solani (1.56), C. albicans (6.2)	[23]
27	T. mentagrophytes (3.91), T. rubrum (3.91), M. canis (1.95), C. albicans (3.91), A. niger (1.95), 3 isolates of S. aureus (3.13), S. pyogenes 308 (6.25), S. pyogenes 77 A (6.25), Staphylococcus. faecium D (6.25)	[21,53,60]
28	Five isolates of C. albicans (1.56~12.5), Trichophyton gypseum, (1.56–15.6), Fusarium graminarum (3.12~7.8), B. subtillis (6.25). 15.6), B. subtilis (31.25)., T. mentagrophytes (3.91–10), T. rubrum (1.95), T. asteroids UC-4775 (1), M. canis (0.48), M. canis UC-1395 (10), Cryptococcus immnzitis UC-1119 (1), C. neoformans UC-1139 (1), A. niger (3.91), A. fumigatus IAM 2046 (12.5), B. subtilis ATCC 6633 (16), B. subtilis UC-564 (4), B. dermatitidlis UC-1911 (1), F. culmorum JP 15 (25), Geotrichutim sp. UC-1207 (1), Hormodendrulmn compactum UC-1222 (1), Hormodendrulmn capsulatum UC-1220 (0.1), Nocardia asteroidles UC-2052 (10), Phialophora verrlucosai UC-1807 (1), 9 isolates of S. aureus (6.25–16), Streptococcus hemolyticus UC- 15 (31), S. pyogenes 308 (25), S. pyogenes 77 A (25), S. faecium D (50), Staphylococcus faecalis UC-3235 (31), 3 isolates of S. epidermidis (32), S. schenlckii UC-1364 (10), 7 isolates of Enterococcus faecalis (32~64), A. fumigatus IAM 2046 (MCC, >200)	[8,21,53,59,60,63,79]
29	T. mentagrophytes (7.81), T. rubrum (7.81), M. canis (1.95), C. albicans (31.2), A. niger (7.81), 3 isolates of S. aureus (50), S. pyogenes 308 (100), S. pyogenes 77 A (100), Penicillium chrysogenum (7.81), B. subtilis (50), Micrococcus luteus (6.25),	[21,53,58,59,60,63,79]
30	Three isolates of S. epidermidis (16–64), 5 isolates of S. aureus (8~16), E. faecalis (64), E. faecium (64), C. albicans ATCC 10231 (16)	[53]
31	Four isolates of S. aureus (64), C. albicans ATCC 10231 (64)	[53]
32	Five isolates of Staphylococcus epidermidis (16~32), S. epidermidis 12-8 (64), C. albicans ATCC 10231 (16)	[53]
33	Five isolates of S. epidermidis (8–32), 2 isolates of S. epidermidis (16~32), E. faecalis ATCC 29212 (32), 2 isolates of E. faecalis (64), 3 isolates of E. faecium 12-3 (64), C. albicans ATCC 10231 (8)	[53]
34	The diameter of inhibition zone (mm): C. albicans (18), F. culmorum JP 15 (18), Glomerella cingulate (13), Klyuveromyces marxianus IMET 25148 (19), P. notatum JP 36 (15), Sporobolomyces salmonicolor SBUG 549 (24)	[60]
35	S. aureus FDA 209P (12.5), C. albicans IAM 4888 (25), S. cerevisiae IAM 4020 (50), A. fumigatus IAM 2153 (50), T. mentagrophytes (12.5), Sporotrichum schenckii (25), Paecilomyces variotii IAM 5001 (6.25), C. albicans (MCC, >200), A. fumigatus (MCC, >200)	[20]
36	S. aureus FDA 209P (50), B. subtillis PCI 219 (50), C. albicans IAM 4888 (100), A. oryzae (25), A. fumigatus (MCC, >200), T. mentagrophytes (6.25), C. albicans (MCC, >200)	[20]
37	S. aureus ATCC 11632 (30), Pseudomonas fluorescens ATCC 13525 (3), Aspergillus viridinutans CBS 12754 (100), P. notatum Tü 136 (30)	[38]
38	Arthrobacter aurescens ATCC 13344 (3), B. subtillis ATCC 6051 (10), S. aureus ATCC 11632 (10), E. coli K12 (10), P. fluorescens ATCC 13525 (0.1), C. albicans ATCC 10231 (10), S. cerevisiae Tü 125 (10), A. viridinutans CBS 12754 (10), P. variotii 137 (30), P. notatum Tü 136 (3)	[38]
39	P. fluorescens ATCC 13525 (3), A. viridinutans CBS 12754 (100), P. notatum Tü 136 (30)	[38]
40	C. albicans (25), A. niger (6.25), P. chrysogenum (7.81), B. subtillis (50), S. aureus (50), M. luteus (6.25), M. canis (1.95), S. pyogenes (100)	[63,64]
41	C. albicans (7), A. niger (3.13), P. chrysogenum (6.25), B. subtillis (15.62), S. aureus (12.50), M. luteus (3.13), M. canis (0.98), S. pyogenes (25)	[63,64]
42 43	B. cinereal Persoon (ID50, 5), Botryosphaeria dothidea (ID50, 1.25); Diameter of inhibition zone (mm): C. neoformans 14 (22), S. cerevisiae 2399 (15), saccharomyces sake yeast (Papulacadnin B resistant strain) (20), C. albicans duke (20), C. albicans duke (Amphotericin B resistant strain) (23), C. albicans 3 (18), C. tropicalis (20), M. gypseum (20), T. mentagrophytes (25), A. niger (12)	[80]
44	S. aureus FDA 209P (12.5), B. subtillis NIHJ PCI 219 (12.5), C. miyabeanus IFO 5277 (3.2), A. mali IFO 8984 (12.5), B. fuckeliana IFO 5363 (1.6), C. lagenarium IFO 7513 (1.6), P. filamentosa sp. Sasakill IFO 6258 (1.6), P. oryzae IFO 5994 (6.25), A. oryzae IFO 5239 (25), T. mentagrophytes IFO 6202 (6.25), S. cerevisiae IFO 0304 (1.6), C. albicans IFO 1594 (1.6)	[66]
46	P. infestans (100), Cladosporium fluvum (25), B. cinereal (25), P. oryzae (25), Cercospora beticola (100)	[67]
49	S. aureus FDA 209P (25), C. albicans Yu 1200 (25)	[51,69]
50	C. albicans Yu1200 (12.5), A. fumigatus IAM 2046(12.5), A. fumigatus (IAM 2046) (MCC, 50)	[58]
51	Four isolates of S. aureus (0.50~1.00)	[70]
52	Four isolates of S. aureus (0.67~1.00)	[70]
53	Four isolates of S. aureus (0.67~0.83)	[70]
54	Four isolates of S. aureus (0.50~0.83)	[70]
55	Four isolates of S. aureus (0.25~0.50)	[71]
56	Four isolates of S. aureus (0.25)	[71]
57	Four isolates of S. aureus (0.25)	[71]
58	S. aureus FDA 209P (12.5), B. subtillis PCI 219 (25), 2 isolates of C. albicans IAM 4888 (25~50), S. cerevisiae IAM 4020 (50), A. fumigatus IAM 2153 (12.5), M. racemosus (12.5), P. variotii IAM 5001(12.5), S. schenckii (12.5), C. albicans Yu 1200 (MCC, >200)	[69]
59	S. aureus FDA 209P (6.25), B. subtillis PCI 219 (12.5), C. albicans IAM 4888 (25), S. cerevisiae IAM 4020 (50), A. fumigatus IAM 2153 (25), M. racemosus (6.25), P. variotii IAM 5001 (12.5), S. schenckii (25)	[69]
60	S. aureus FDA 209P (12.5), B. subtillis PCI 219 (25), C. albicans IAM 4888(25), S. cerevisiae IAM 4020 (50), A. fumigatus IAM 2153 (50), M. racemosus (25), P. variotii IAM 5001 (12.5), S. schenckii (25)	[69]
61	S. aureus FDA 209P (6.25), B. subtillis PCI 219 (12.5), C. albicans IAM 4888 (25), S. cerevisiae IAM 4020 (50), A. fumigatus IAM 2153 (25), M. racemosus (12.5), P. variotii IAM 5001 (12.5), S. schenckii (25)	[69]
62	S. aureus FDA 209P (0.78), B. subtillis PCI 219 (1.56), 3 isolates of C. albicans (3.12~25), S. cerevisiae IAM 4020 (6.25), A. fumigatus IAM 2153 (3.12), M. racemosus (3.12), P. variotii IAM 5001 (1.56), S. schenckii (3.12)	[69]
63	T. mentagrophytes (15.6), T. rubrum (15.6), M. canis (7.81), C. albicans (6.25~31.2), A. niger (15.6), A. fumigatus IAM 2046 (6.25), 3 isolates of S. aureus 6511 (12.5), S. pyogenes 308 (6.25), S. pyogenes 77 A (6.25), S. faecium D (50), A. fumigatus (MCC, 12.5)	[21,58,60,53]

Azalomycin F has remarkable bioactivities against Gram-positive bacteria, yeast, fungi, and protozoa [17,34,73,75], and some clinical studies on its anti-trichomoniasis and anti-candida infectious effects were performed [34], while no corresponding drug was approved in the clinic. Stefanelli et al. discovered that niphimycin (28) and azalomycin F (8–10) could inhibit the type-I interleukin-1 receptor [55]. Moreover, they had remarkable activities against phytopathogenic fungi, such as Fusarium oxysporum, Fusarium moliniforme, Asparagus officinalis, Rhizoctonia solani, Colletotrichum gloeosporioides, and Alternaria mali [17,36].

Hamagishi et al. [48] discovered that copiamycin A (1), azalomycin F (8–10), and scopafungin (niphimycin, 28) could inhibit the secretion of gastric acid in the gastric parietal cells of rats by inhibiting H+/K+-ATPase with the IC50s of 15.7, 16.4, and 35.9 μg/mL, respectively. The inhibitory potency of copiamycin was found to be comparable to that of omeprazole and SCH-28080, both specific inhibitors of the gastric H+/K+-ATPase in vitro and in vivo [48].

In addition, Reusser [56] proposed that niphimycin (scopafungin, 28) was an inhibitor of mitochondrial oxidative phosphorylation and respiration, and mainly a decoupling agent for oxidative phosphorylation. Furthermore, Mogi et al. [81] discovered that niphimycin had inhibitory activity against NADH dehydrogenase (NDH-II), and deduced that niphimycin had a great potential to become an antibacterial drug as it showed no severe effect on mammalian respiratory enzymes.

Using an NIH3T3 cell line, a screening system for Ras signal inhibitors was developed to search for anti-cancer agents by Futamura et al. [74]. Malolactomycin D (47) was identified as a selective inhibitor of Ras-responsive transcription. The expression of matrix metalloproteinases MMP-1 and MMP-9 in NIH3T3 cells line could be reduced by treatment with malolactomycin D at the translational and transcriptional levels, and this was achieved likely by inhibiting the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) [82,83]. As MMPs contribute to tumor growth, invasion, and metastasis by promoting the degradation of extracellular matrix and maintaining the tumor microenvironment [84,85], malolactomycin D suppressing the transformation activity of Ras-transformed cells by inhibiting the expression of Ras-inducible genes, such as MMP-1 and MMP-9, indicated that it was expected to be a new anticancer agent with high efficiency and low toxicity.

Ko et al. [40] isolated a phospholipase C inhibitor (PLC) from the culture medium of actinomycete MT2617-2 and named it as MT2617-2B, which produced its two isomers having the same molecular weight by standing in methanol solution at room temperature, copiamycin and niphithricin A. Besides antimicrobial activities against S. aureus and C. albicans, MT2617-2B had a remarkable inhibitory activity against phospholipase C with the IC50 values against PLC-γ1 and PLC-β1 of 25 and 50 μg/mL, respectively.

5. Acute Toxicity

To understand the safety of these compounds, the median lethal dose (LD50) and maximal tolerable dose (MTD or LD0) of some guanidine-containing polyhydroxyl macrolides were determined. As shown in Table 4, the LD50 or LD0 doses of each compound successively decreased from oral, subcutaneous, intraperitoneal to intravenous administrations. Moreover, Benziger and Edelson reported that azalomycin F administered intravaginally presented limited absorption [86]. Thereby, we deduced that azalomycin F administered orally was likely difficult to be absorbed, and this might be in accordance with the experimental results of its acute toxicities in different administrations. It was inexplicable that the LD50 or LD0 of neocopiamycins A and B administered intraperitoneally were more than 1000 mg/kg, which indicated that they had low toxicity, while the LD50 or LD0 of neocopiamycins A and B administered intravenously were only more than 30 or 25 mg/kg. Moreover, these compounds, except for neocopiamycins A and B in Table 4, had similar acute toxicities when they were administrated intraperitoneally. This indicated that their toxicities might be attributed to the lactone ring and guanidyl side-chain, which were also mainly responsible for their antimicrobial activities, and likely had nothing to do with the size of lactone ring and the numbers of hydroxyl and methyl groups. It was worth noting that the purities of compounds or mixtures used for the acute toxicity test would fluctuate the experimental results; however, very few publications have provided this information.

Table 4

The acute toxicities of some guanidine-containing polyhydroxyl macrolides.

Compounds	Organisms	Test Type	Administration	Dose (mg/kg)	References
Copiamycin	mouse	LD50	Intraperitoneal	24.8	[76]
	mouse	LD50	Subcutaneous	61.5	[76]
Neocopiamycins A and B	mouse	LD0	Intraperitoneal	>1000	[50]
	mouse	LD0	Intravenous	>30 and >25	[50]
	mouse	LD0	Oral	>1000	[50]
Azalomycin F	mouse	LD50	Intraperitoneal	18 or 26	[87,88]
	mouse	LD50	Intravenous	12.5	[73]
	mouse	LD50	Oral	580	[73]
	mouse	LD50	Subcutaneous	162	[73]
Azalomycin F a	mouse	LD50	Intraperitoneal	97.9	[25]
Guanidylfungin A	mouse	LD50	Intraperitoneal	12.5	[20]
Malolactomycin A	mouse	LD50	Intraperitoneal	6.7	[66]
Malolactomycin C	mouse	LD0	Intraperitoneal	>30	[67]
Malolactomycin D	mouse	LD0	Intraperitoneal	>30	[67]
RS-22 b	mouse	LD50	Intravenous	25	[38]

a: a mixture of twelve azalomycin F analogs was used in the determination of LD50. b: a mixture of RS-22 A, B, and C was used in the determination of LD50.

6. Antimicrobial Mechanisms

As these compounds had remarkable inhibitory activities against Gram-positive bacteria and fungi, related researches mainly focused on antibacterial and antifungal mechanisms. Previous works indicated that cell membrane was the main action site of them against bacteria and fungi, and these compounds could change the plasma membrane permeability and lead to the leakage of cellular substances [26,27,28,30].

6.1. Antibacterial Mechanisms

As Sugawara reported [75], azalomycin F could lead to the leakage of cellular substances to kill Bacillus subtilis, while detailed mechanisms had not been further reported because their chemical structures were not clear at that time. Inspired by the fact that the antimicrobial activities of azalomycin F and copiamycin could be reversed in the same manner by the phospholipid fraction of the bacteria, and various phospholipids, such as phosphatidylglycerol (PG) and phosphatidylcholine [89], Yuan et al. [26] discovered that azalomycin F5a, the main component of azalomycin F, could lead to the leakage of cellular substances possibly by increasing permeability to kill S. aureus and confirmed that cell-membrane lipids, especially 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG), might be important targets of azalomycin F5a against S. aureus after its relative configurations were assigned [24,26]. Further researches indicated that azalomycin F5a, increasing the cell membrane permeability of S. aureus, was likely achieved by the synergy of its lactone ring binding to the polar head of phospholipid and its guanidyl side-chain targeting to lipoteichoic acid (LTA), and which had eventually led to the autolysis of S. aureus cells [30]. The compositional analysis indicated that PG, lysyl-phosphatidylglycerol (LPG), and cardiolipin (CL) were three major components of S. aureus cell-membrane phospholipid, and PG was the largest one [90,91]. Simultaneously, the content of lysyl-DPPG in the cell-membrane lipids would increase when S. aureus was resistant to daptomycin [90]. Thereby, molecular dynamics simulation, showing that azalomycin F5a had greater adhesive force to plasma membrane assembled by DPPG plus lysyl-DPPG than by DPPG, indicated that azalomycin F5a likely had greatly antagonistic activity to daptomycin-resistant S. aureus strains, and then proposed that these compounds had a great potency to be developed into new antimicrobial agents as LTA is also an important target for new antibiotics [92,93].

6.2. Antifungal Mechanism

Although these compounds can change the cell membrane permeability of microbe and lead to the leakage of cellular substances, there are different mechanisms of them against bacteria and fungi as the components of their cell envelopes are different.

Sugawara [94] discovered that azalomycin F could cause the leakage of cellular substance from the cells of C. albicans and the lysis of rabbit erythrocytes, and strongly inhibit amino acid incorporation into cellular protein and oxidative deamination of amino acid metabolism, but not decarboxylation and transamination. Simultaneously, it insignificantly inhibited the incorporation of phosphate into nucleic acids and the glycolytic pathway and did not exert any noticeable inhibition in cell-free protein-synthesizing systems of E. coli, rat liver, and C. albicans, and mitochondrial enzyme systems. Thereby, the cell surface was proposed as the primary site of azalomycin F acting on C. albicans [94]. Moreover, antifungal mechanisms of other guanidine-containing polyhydroxyl macrolides also confirmed that these compounds, such as niphimycins and copiamycins, could act on the cell membrane of fungi and alter their membrane permeability to cause the leakage of cellular components [27,28,89]. Further researches proposed that copiamycin and zalomyci F disrupted the cell membrane of fungi by binding to the cell-membrane phospholipids [89]. Thorough antifungal mechanism indicated that a synergistic combination of direct plasma membrane damage and oxidative stress was a cause of antifungal activity of niphimycin against Saccharomyces cerevisiae [29], and proposed that niphimycin disrupted the plasma membrane by directly interacting with phospholipids, such as phosphatidylcholine, but did not interact with ergosterol, a molecular target of amphotericin B. At the same time, Nakayama et al. [29], depending on the differences in the structures of niphimycin and amphotericin, suggested that the ability of niphimycin damaging the plasma membrane and/or generating ROS residues was primarily attributed to the alkyl side chain and terminal guanidine. In addition, Uno et al. [77] inferred that copiamycin, a 32-membered guanidyl polyol macrolide, had ionophoretic activity and could form a conformation with a ring or cavity that focuses the oxygens in lactone ring with various cations into a complex.

7. Antimicrobial Structure-Activity Relationship

As antimicrobial activity is one of the most important bioactivities of these compounds, most of them presented their minimum inhibitory concentration (MIC) against bacteria and fungi (Table 3) when they were discovered. Thereby, the structure-activity relationships of these compounds against bacteria and fungi can be summarized as follows:

(1) The atom number composed of the lactone ring is less important for their antimicrobial activities [50,66,86] and acute toxicity (Table 4). It is worth noting that 32-membered guanidine-containing polyhydroxyl macrolide TCM-34 shows remarkable antifungal activity (MIC, 1.6 to 3.1 μg/mL), while presents very weak antibacterial activity (MIC, more than 100 μg/mL) [52].

(2) Antimicrobial activity is significantly affected by the guanidyl side-chain, especially by the terminal guanidine group, which is a key for their antibacterial and antifungal activities. The substitution of guanidino residue to urea will lead to the loss of antibacterial activity and significantly narrow the antifungal spectrum [29,38,95], while the number of methyl groups linking on guanidine has a little or no effect on the antimicrobial activity [12,18,20]. Moreover, enough length (9 or 11 carbons) of the side chain is necessary for the antimicrobial bioactivity [96].

(3) The hydrolysis of the lactone ring will lead to the loss of antimicrobial activity [95]. The six-membered hemiketal ring plays an essential role in the antimicrobial activity, and the opening of a six-membered hemiacetal ring will remarkably decrease the antimicrobial activity [50]. Simultaneously, the etherification of C17 hydroxyl will slightly reduce the antimicrobial activity, and sometimes this decrease can be counteracted by the increase of antimicrobial activity due to the removal of the malonyl group [21,50,58,70,71].

(4) There is no significant influence on the antimicrobial activity when hydrogenation, methyl removal, or/and methyl substitution occur to the double bond of the lactone ring. Similarly, methyl substitution of the double bond on the guanidyl side-chain is less important for the antimicrobial activity [20,23,38,63].

(5) The introduction of malonyl moiety will reduce the antimicrobial activity [21,50,53,70,71,97]. The more the number of malonyl substitution, the weaker the antibacterial activity of these compounds. However, the position of malonyl substitution shows no influence on their antibacterial activities [53].

As we reported [30], azalomycin F5a could increase the cell membrane permeability of S. aureus and eventually lead to the autolysis of S. aureus cells, by the synergy of its lactone ring binding to the polar head of phospholipid and its guanidyl side-chain targeting to LTA, which is a vital anion component anchoring on the phospholipid bilayer of Gram-positive bacteria. This was in accordance with the above structure-activity relationship that the lactone ring and the terminal guanidyl side-chain were vital for the antimicrobial activity. As the carboxyl group of malonyl monoester can theoretically form an intramolecular hydrogen bond or ionic bond with the guanidyl of side-chain, the existence of malonyl will likely block the interaction between the guanidyl of side-chain and LTA. This was confirmed by their 3D molecular structures obtained by ChemBio3D Ultra 12.0 run with MM2 calculation (Figure 6) and by pharmacophore model of 36-membered guanidine-containing polyhydroxyl macrolides using Discovery Studio 3.5 (Figure 7), and could explain why the antimicrobial activity of azalomycin F was greatly reduced by phospholipids containing an acidic phosphoryl group [98]. Further, this can also explain why the introduction of malonyl will greatly reduce the antimicrobial activity and coincides with the above structure-activity relationship (5). From Figure 5, we can deduce that the substituted position (C-19, C-23, or C-25) of malonyl coincides with the spatial distance of the intramolecular salt or hydrogen bond formation between the terminal guanidine and the carboxyl group of malonyl monoester. This will likely reduce the interaction between the guanidyl of side-chain and LTA, and then reduce the antimicrobial activity of these compounds. Inspired by a previous publication [99], all these above indicate that the introduction of malonyl may be self-protection of actinomycetes producing guanidine-containing polyhydroxyl macrolide, through which they can be free from the poison and injury of secondary metabolites produced by themselves. Moreover, the demalonylation of these compounds not only increases the antimicrobial activity but also yields a basic compound, which has a better water solubility, especially for its hydrochloride [50,71].

Figure 6

The 3D molecular structures of azalomycin F5a (a) and 23-demalonyl azalomycin F5a (b) obtained by ChemBio3D Ultra 12.0. (a) An intramolecular hydrogen bond or ionic bond (dotted line) is formed between the guanidyl (nitrogen atoms colored blue) of side-chain and the carboxyl group (oxygen atoms colored red) of malonyl monoester, but there is no bond formation in case of (b).

Figure 7

Pharmacophore model of 36-membered guanidine-containing polyhydroxyl macrolides obtained by Discovery Studio 3.5. Ten pharmacophore features were constructed, and was successively F1:Acc|Don (Hydrogen bond acceptor or donor); F2:Acc (Hydrogen bond acceptor); F3, F4, F6, and F7:Hyd (Hydrophobic region); F5:Don&Acc (Hydrogen bond donor and acceptor); F8 Acc2|Don2 (Hydrogen bond acceptor or donor projected); and F9 and F10:Don2&Acc2 (Hydrogen bond donor and acceptor projected).

8. Conclusions

To date, a total of 63 guanidine-containing polyhydroxyl macrolides were reported, including 48 prototype compounds isolated from 33 actinomycete strains and 15 structural derivatives. These compounds have various bioactivities, such as broad-spectrum antimicrobial activity, anti-trichomonas, anti-tumor, and inhibitory activities against H+/K+-ATPase, mitochondrial oxidative phosphorylation, NADH dehydrogenase, and phospholipase C, while they also have a little toxicity. Structure-activity relationships indicate that both the terminal guanidine group and the lactone ring are the key for their antimicrobial activities. As LTA anchoring to the cell membrane is an important polymer for the resistance to cationic antibiotics, the autolysin regulation, and the cell division of Gram-positive bacteria, LTA synthase gradually becomes a proposed drug target for the development of antibiotics against drug-resistant Gram-positive bacteria [92,93,100,101]. Thereby, the discovery of guanidyl side-chain targeting to lipoteichoic acid indicates these compounds have a great potency to be developed into antimicrobial and anti-inflammatory drugs.