Thiophenes-Naturally Occurring Plant Metabolites: Biological Activities and In Silico Evaluation of Their Potential as Cathepsin D Inhibitors.

Naturally, thiophenes represent a small family of natural metabolites featured by one to five thiophene rings. Numerous plant species belonging to the family Asteraceae commonly produce thiophenes. These metabolites possessed remarkable bioactivities, including antimicrobial, antiviral, anti-inflammatory, larvicidal, antioxidant, insecticidal, cytotoxic, and nematicidal properties. The current review provides an update over the past seven years for the reported natural thiophene derivatives, including their sources, biosynthesis, spectral data, and bioactivities since the last review published in 2015. Additionally, with the help of the SuperPred webserver, an AI (artificial intelligence) tool, the potential drug target for the compounds was predicted. In silico studies were conducted for Cathepsin D with thiophene derivatives, including ADMET (drug absorption/distribution/metabolism/excretion/and toxicity) properties prediction, molecular docking for the binding interaction, and molecular dynamics to evaluate the ligand-target interaction stability under simulated physiological conditions.

1. Introduction

Heterocyclic compounds display a remarkable role in the field of bioactive metabolites search. It is noteworthy that >75% of clinically utilized drugs possess heterocyclic moiety in their chemical skeleton [1]. Sulfur belongs to chalcogens that are the 16 group elements of the periodic table. Sulfur is a ubiquitous heteroatom in medicinal chemistry that can bond to various atoms, including nitrogen, oxygen, carbon, halides, and phosphorus. Several sulfur-based functionalities have become privileged pharmacophores in synthesizing new derivatives that contribute to drug discovery [2]. In living organisms, it displays a remarkable characteristic of possessing a variety of redox potentials and redox states, producing many sulfur species that take part in diverse biological processes. Thioethers and thiols can form sulfonium ions by donating electrons to other organic species, revealing their ability to stabilize a negative charge on a neighboring carbon [3]. They can undergo sequential oxidation to sulfoxides and sulfones, which have diverse biological roles. For example, S-adenosylmethionine (SAM—sulfonium compound) mediates most biochemical methylation reactions in cell metabolism [4].

S-containing species have featured a strong electron-withdrawing nature, resistance to reduction at sulfur, stability against hydrolysis, and preference for two electrons over radical processes that make this group of compounds applicable to many drug research fields [5]. Their diverse pharmacological potential makes it the first choice for incorporation by the hybrid approach, which is present in most of the required medicines accessible in the market [5]. It was reported that 41 sulfur-containing commercial drugs appeared in the Top 200 Pharmaceuticals by Retail Sales in 2019 worldwide; 20.5% contain a sulfur atom [6].

Natural products have attracted significant attention as a potential source of S-containing compounds for drug discovery. The well-known conotoxin, ecteinascidin 743 (ET-743), and penicillin are examples of natural sulfur-containing clinical drugs. Furthermore, many sulfur-containing drugs are derived from natural products, e.g., phthalascidin and ixabepilone for cancer treatments, rosuvastatin for hyperlipidemia, and dalfopristin and quinupristin for infectious diseases [7].

Thiophenes are among the heterocyclics that have been located in the focus of research interest for the last decades. They are a class of sulfur-containing molecules usually composed of one to five thiophene units connected at the α-position and often have various alkyl groups at the α’-carbon of the terminal ring [8]. Thiophene derivatives have beneficial applications in the dye, pharmaceutical, and agrochemical industries [9,10]. Interestingly, many of the approved drugs available in the markets have thiophene moiety, including antiasthma, NSAIDs (non-steroidal anti-inflammatory drugs), diuretics, anticancer, and antihistaminic drugs [11,12]. Natural occurring thiophenes represent rare constituents reported from these metabolites that have been isolated from various Asteraceae genera: Echinops, Eclipta, Pluchea, Artemisia, Tagetes, Porophyllum, Atractylodes, Atractylodes, and Xanthium. Additionally, some are reported from Ferula (family Apiaceae), as well as from actinomycetes (Streptomyces) and fungi (Penicillium) (Figure 1) [8].

Figure 1

Number of reported thiophenes from various sources.

They are produced as a chemical defense mechanism and are toxic to various pathogens, such as insects, nematodes, bacteria, and fungi [13,14]. Biosynthetically, they are derived from fatty acids or polyacetylenes through acetylene intermediates; therefore, they are named acetylenic thiophenes. Indeed, many of the reported derivatives possess an alkyl chain with an acetylenic unit that may contain chiral centers due to introducing a hydroxy group [8]. These metabolites have remarkable biological and pharmacological effectiveness, including antiviral, antimicrobial, antileishmanial, anti-inflammatory, larvicidal, antioxidant, insecticidal, HIV-1 (human immunodeficiency virus-1) protease inhibitory, cytotoxic, nematicidal, and phototoxic effects [8,15,16,17,18,19,20] (Figure 2).

Figure 2

Biological activities of thiophenes.

In our previous review, 96 natural thiophene derivatives were listed from various plant species belonging to the Asteraceae family till 2015, with a particular focus on their biosynthesis, bioactivities, and physical and spectral data [8]. Recently, several reviews dealing with synthetic thiophene-based derivatives, including their anti-inflammation and anticancer potentials, spectroscopic properties, and synthesis, were published [21,22,23,24]. On the other side, there is no available review on naturally occurring thiophene derivatives from plant sources.

Therefore, the current review aims to provide an update over the past seven years for the naturally reported thiophene derivatives, including their sources, biosynthesis, spectral data, and bioactivities. In total, 96 compounds have been listed that have been categorized according to the number of rings into mono-, bi-, ter, and quinque-thiophenes and miscellaneous derivatives. Additionally, their source, molecular weights and formulae, location, and fraction/extract from which they were isolated are listed in Table 1. The physical constants and spectral data of the newly reported thiophenes (Table S1) from 2015 to 2021 are included. Further, their possible biosynthetic pathways are illustrated in Scheme 1 and Scheme 2 and bioactivities are highlighted in Table 2. We hope that this review can help natural product researchers for structural characterization of these metabolites and direct the medicinal chemist to the synthesis of potentially more active new thiophene derivatives. A systematic search for the published data was performed in various databases, including Web of Science, PubMed, Scopus, and Google scholar. Moreover, published papers in different publishers such as ACS (American Chemical Society), Elsevier, Bentham, Sage, Wiley, Taylor & Francis, Thieme Medical, and Springer were surveyed. No language restrictions were applied.

Table 1

Naturally occurring mono-, bi-, ter-, and quinquethiophenes and miscellaneous derivatives (name, source, molecular weights and formulae, and location).

Compound Name	Source	Extract/Fraction	Mol. Wt.	Mol. Formula	Location	Ref.
I. Monothiophenes
Foetithiophene A (1)	Ferula foetida, roots (Apiaceae)	MeOH/EtOAc-soluble fraction	170	C8H10O2S	Dorouneh village mountains, Khorasan Razavi, Iran	[25]
Foetithiophene B (2)	Ferula foetida, roots (Apiaceae)	MeOH/EtOAc-soluble fraction	218	C9H14O2S2	Dorouneh village mountains, Khorasan Razavi, Iran	[25]
Foetithiophene C (3)	Ferula foetida, roots (Apiaceae)	MeOH/EtOAc-soluble fraction	186	C9H14S2	Dorouneh village mountains, Khorasan Razavi, Iran	[25]
Foetithiophene D (4)	Ferula foetida, roots (Apiaceae)	MeOH/EtOAc-soluble fraction	310	C16H22S3	Dorouneh village mountains, Khorasan Razavi, Iran	[25]
Foetithiophene E (5)	Ferula foetida, roots (Apiaceae)	MeOH/EtOAc-soluble fraction	202	C9H14OS2	Dorouneh village mountains, Khorasan Razavi, Iran	[25]
Foetithiophene F (6)	Ferula foetida, roots (Apiaceae)	MeOH/EtOAc-soluble fraction	188	C9H16S2	Dorouneh village mountains, Khorasan Razavi, Iran	[25]
5-Propinyl-thiophene-2-Carboxylic acid = Junipic acid (7)	Artemisia sieversiana, aerial parts (Asteraceae)	EtOH/CH3Cl-soluble fraction	166	C8H6O2S	Qinghai, China	[26]
	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	-	-	Qinghe County, Xinjiang, China	[27]
3-Hydroxy-5-propinyl-2-acetyl-thiophene (8)	Artemisia sieversiana, aerial parts (Asteraceae)	EtOH/CH3Cl-soluble fraction	180	C9H8O2S	Qinghai, China	[26]
2-(3,4-Dihydroxybut-1-ynyl)-5-(penta-1,3-diynyl)thiophene = 2-(Penta-1,3-diynyl)-5-(3,4-dihydroxybut-1-ynyl)-thiophene =4-(5-(Penta-1,3-diyn-1-yl)thiophen-2-yl)but-3-yne-1,2-diol = 2-(Penta-1,3-diynyl)-5-(3,4-dihydroxybut-1-ynyl)-thiophene = 5-(Penta-1,3-diynyl)-2-(3,4-dihydroxybut-1-ynyl)-thiophene (9)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	230	C13H10O2S	Nantou, Taiwan	[28]
	Echinops giganteus, roots (Asteraceae)	MeOH/CH2Cl2-soluble fraction	-	-	Dschang, Western Region of Cameroon	[29]
	Echinops giganteus, rhizomes and aerial parts (Asteraceae)	MeOH/n-hexane-soluble fraction	-	-	Dschang, Western Region of Cameroon	[30]
	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	-	-	Qinghe County, Xinjiang, China	[27]
2-(4-Hydroxybut-1-ynyl)-5-(penta-1,3-diynyl)thiophene (10)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	214	C13H10OS	Nantou, Taiwan	[28]
2-(Penta-1,3-diyn-1-yl)-5–(4-acetoxy-3-hydroxybuta-1-yn-1-yl) thiophene (11)	Pluchea indica, aerial parts (Asteraceae)	EtOH/n-hexane-soluble fraction	256	C15H12O2S	Bon subdistrict, Khlung, Chantaburi, Thailand	[31]
5-(Penta-1,3-diynyl)-2-(3-hydroxy-4-acetoxybut-1-ynyl)-thiophene (12)	Echinops hoehnelii, roots (Asteraceae)	MeOH/CH2Cl2-soluble fraction	272	C15H12O3S	Bale Mountains National Park, Ethiopia	[32]
5-(Penta-1,3-diynyl)-2-(3-methoxy-4-acetoxy-but-1-yn)-thiophene (13)	Echinops hoehnelii, roots (Asteraceae)	MeOH/CH2Cl2-soluble fraction	286	C16H14O3S	Bale Mountains National Park, Ethiopia	[32]
5-(Penta-1,3-diynyl)-2-(3-chloro-4-acetoxy-but-1-yn)-thiophene (14)	Echinops hoehnelii, roots (Asteraceae)	MeOH/CH2Cl2-soluble fraction	290	C15H11ClO2S	Bale Mountains National Park, Ethiopia	[32]
Echinothiophene A (15)	Echinops grijisii, roots (Asteraceae)	EtOH/CH2Cl2-soluble fraction	248	C13H9ClOS	Zhangjiakou Hebei, China	[19]
Echinothiophene B (16)	Echinops grijisii, roots (Asteraceae)	EtOH/CH2Cl2-soluble fraction	290	C15H11ClO2S	Zhangjiakou Hebei, China	[19]
Echinothiophene C (17)	Echinops grijisii, roots (Asteraceae)	EtOH/CH2Cl2-soluble fraction	398	C23H26O4S	Zhangjiakou Hebei, China	[19]
2-(Pro-1-ynyl)-5-(5,6-dihydroxypenta-1,3-diynyl) thiophene (PYDDT) = 2-(Prop-1-ynyl)-5(5,6-dihydroxyhexa-1, 3-diynyl)-thiophene = PITC-2 = R/J/3 (18)	Echinops grijisii, roots (Asteraceae)	EtOH/CH2Cl2-soluble fraction	230	C13H10O2S	Bozhou north of Anhui, China	[33]
	Pluchea indica, aerial parts (Asteraceae)	EtOH/n-hexane-soluble fraction	-	-	Bon subdistrict, Khlung, Chantaburi, Thailand	[31]
5-(1,2-Dihydroxyethyl)-2-(E)-hept-5-ene-1,3-diynylthiophene (19)	Echinops latifolius, roots (Asteraceae)	MeOH/CH2Cl2-soluble fraction	232	C13H12O2S	Seoul, Korea	[34]
5-(1,2-Dihydroxy-ethyl)-2-(Z)-hept-5-ene-1,3-diynylthiophene (20)	Echinops latifolius, roots (Asteraceae)	MeOH/CH2Cl2-soluble fraction	232	C13H12O2S	Seoul, Korea	[34]
5-(Penta-1,3-diynyl)-2-(3-methoxy-4-hydroxybut-1-ynyl)-thiophen (21)	Echinops hoehnelii, roots (Asteraceae)	MeOH/CH2Cl2-soluble fraction	244	C14H12O2S	Bale Mountains National Park,Ethiopia	[32]
2-(Prop-1-inyl)-5-(6-acetoxy-5-hydroxyhexa-1,3-diinyl) thiophene (22)	Pluchea indica, aerial parts (Asteraceae)	EtOH/n-hexane-soluble fraction	256	C15H12O2S	Bon subdistrict, Khlung, Chantaburi, Thailand	[31]
53′′R-Pluthiophenol (23)	Pluchea indica, aerial parts (Asteraceae)	EtOH/n-hexane-soluble fraction	230	C13H10O2S	Hepu, Guangxi, China	[35]
3′′R-Pluthiophenol-4′′-acetate (24)	Pluchea indica, aerial parts (Asteraceae)	EtOH/n-hexane-soluble fraction	272	C15H12O3S	Hepu, Guangxi, China	[35]
3′′-Ethoxy-3′′S-pluthiophenol (25)	Pluchea indica, aerial parts (Asteraceae)	EtOH/n-hexane-soluble fraction	258	C15H14O2S	Hepu, Guangxi, China	[3]
3′′-Ethoxy-3′′S-pluthiophenol-4′′-acetate (26)	Pluchea indica, aerial parts (Asteraceae)	EtOH/n-hexane-soluble fraction	300	C17H16O3S	Hepu, Guangxi, China	[35]
Rupestriene B (27)	Artemisia rupestris, whole plant (Asteraceae)	EtOH/CH3Cl-soluble fraction	234	C13H14O2S	Xinjiang Uygur Autonomous,China	[36]
Rupestriene C (28)	Artemisia rupestris, whole plant (Asteraceae)	EtOH/CH3Cl-soluble fraction	234	C13H14O2S	Xinjiang Uygur Autonomous,China	[36]
Atracthioenyneside A (29)	Atractylodes lancea, rhizomes (Asteraceae)	EtOH/n-BuOH extract	414	C19H26O8S	Huanggang, Hubei, China	[37]
Atracthioenyneside B (30)	Atractylodes lancea, rhizomes (Asteraceae)	EtOH/n-BuOH extract	386	C17H22O8S	Huanggang, Hubei, China	[37]
(Z)-6-(5-(Prop-1-yn-1-yl)thiophen-2-yl)hex-3-en-5-yne-1,2-diol (31)	Eclipta prostrata, aerial parts (Asteraceae)	EtOH/PE-soluble fraction	232	C13H12O2S	Mount Kunyu area, Shandong, China	[38]
II. Bithiophenes
5-(4-Hydroxy-3-methoxy-1-butyny)-2,2′-bithiophene (32)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	264	C13H12O2S2	Nantou, Taiwan	[28]
5-(3,4-Dihydroxybut-1-ynyl)-2,2′-bithiophene = 4-([2,2′-bithiophen]-5-yl) but-3-yne-1,2-diol (33)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	250	C12H10O2S2	Nantou, Taiwan	[28]
	Eclipta prostrata, aerial parts (Asteraceae)	MeOH/EtOAc-soluble fraction	-	-	Seoul, Korea	[39]
	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	-	-	Qinghe County, Xinjiang, China	[27]
5-Acetyl-2,2′-bithiophene = Ethanone (34)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	208	C10H8OS2	Nantou, Taiwan	[28]
	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	-	-	Qinghe County, Xinjiang, China	[27]
5-Formyl-2,2′-bithiophene (35)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	194	C9H6OS2	Nantou, Taiwan	[28]
Methyl 2,2′-bithiophene-5-carboxylate (36)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	224	C10H8O2S2	Nantou, Taiwan	[28]
5-(But-3-en-1-ynyl)-2,2′-bithiophene (5-BBT) (37)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	216	C12H8S2	Nantou, Taiwan	[28]
	Porophyllum obscurum, aerial parts (Asteraceae)	n-Hexane extract	-	-	Las Chacras, province of San Luis, Argentina	[40]
	Echinops grijisii, roots (Asteraceae)	Essential oil, hydrodistillation	-	-	Nanjing, Jiangsu, China	[41]
	Echinops albicaulis, aerial parts (Asteraceae)	MeOH/CH2Cl2 soluble fraction	-	-	Malaysary gorge, Kazakhstan	[42]
5-(4-Isovaleroyloxybut-1-ynyl)-2,2′-bithiophene (5-IBT) (38)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	318	C17H18O2S2	Nantou, Taiwan	[28]
	Echinops grijisii, roots (Asteraceae)	Essential oil, hydrodistillation	-	-	Nanjing, Jiangsu, China	[41]
Cardopatine (39)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	432	C24H16S4	Nantou, Taiwan	[28]
Isocardopatine (40)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	432	C24H16S4	Nantou, Taiwan	[28]
5-(3-Hydroxy-4-isovaleroyloxybut-1-ynyl)-2,2′-bithiophene (41)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	334	C17H18O3S2	Nantou, Taiwan	[28]
5-(3-Hydroxymethyl-3-isovaleroyloxyprop-1-ynyl)-2,2′-bithiophene (42)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	334	C17H18O3S2	Nantou, Taiwan	[28]
5-(4-Hydroxy-1-butynyl)-2,2′-bithiophene = 4-([2,2′-bithiophen]-5-yl)but-3-yn-1-ol = 4-(5′-(hydroxymethyl)-[2,2′-bithiophene]-5-yl)but-3-yn-1-ol) (43)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	234	C12H10OS2	Nantou, Taiwan	[28]
	Porophyllum obscurum, aerial parts (Asteraceae)	n-Hexane extract	-	-	Las Chacras, province of San Luis, Argentina	[40]
	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	-	-	Qinghe County, Xinjiang, China	[27]
	Tagetes minuta, aerial parts (Asteraceae)	MeOH/n-hexane-soluble fraction	-	-	Al-Baha city, Saudi Arabia	[43]
5-(4-Acetoxy-1-butynl)-2,2′-bithiophene (44)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction			Nantou, Taiwan	[28]
	Porophyllum obscurum, aerial parts (Asteraceae)	n-Hexane extract	-	-	Las Chacras, province of San Luis, Argentina	[40]
2,2′-Bithiophene-5-carboxylic acid (45)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	209	C9H6O2S2	Nantou, Taiwan	[28]
	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	-	-	Qinghe County, Xinjiang, China	[27]
Thiotagetin B: [(Z)-1′′-([2,2′-bithiophen]-5-yl)-8′′-chloro-6′′,11′′-dimethylundeca-6′′,10′′-dien-2′′-yn-9′′-one] (46)	Tagetes minuta, aerial parts (Asteraceae)	MeOH/n-hexane-soluble fraction	388	C21H21ClOS5	Al-Baha city, Saudi Arabia	[44]
1-([2,2′-Bithiophen]-5-yl)-4 -hydroxybutan-1-one (47)	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	252	C12H12O2S2	Qinghe County, Xinjiang, China	[27]
6-Methoxy-arctinol-b (48)	Echinops latifolius, roots (Asteraceae)	MeOH/CH2Cl2-soluble fraction	278	C14H14O2S2	Seoul, Korea	[34]
Arctinol-b (49)	Echinops latifolius, roots (Asteraceae)	MeOH/CH2Cl2-soluble fraction	264	C13H12O2S2	Seoul, Korea	[34]
	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	-	-	Qinghe County, Xinjiang, China	[27]
Arctinone-b (50)	Echinops grijisii, roots (Asteraceae)	EtOH/CH2Cl2-soluble fraction	246	C13H10OS2	Zhangjiakou Hebei, China	[19]
Arctinol (51)	Echinops latifolius, roots (Asteraceae)	MeOH/CH2Cl2-soluble fraction	248	C12H8O2S2	Seoul, Korea	[34]
Arctinal (52)	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	232	C12H8OS2	Qinghe County, Xinjiang, China	[27]
Arctinol A (53)	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	234	C12H10OS2	Qinghe County, Xinjiang, China	[27]
Arctic acid (54)	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	248	C12H8O2S2	Qinghe County, Xinjiang, China	[27]
Methyl [5′-(1-propynyf)-2,2′-bithienyl-5-yl] carboxylate (55)	Echinops latifolius, roots (Asteraceae)	MeOH/CH2Cl2-soluble fraction	262	C13H10O2S2	Seoul, Korea	[34]
2,2-Dimethyl-4-[5′-(prop-1-ynyl)-2,2′-bithiophen-5-yl]-1,3-dioxolane (56)	Echinops spinosissimus subsp. Spinosus, roots (Asteraceae)	EtOH/CH2Cl2-soluble fraction	304	C16H16O2S2	Morocco	[45]
5′-(3,4-Dihydroxybut-1-yn-1-yl)-[2,2′-bithiophene]-5-carbaldehyde = 5-[l-(4-hydroxybut-l-ynyl)]-2,2′-bithiophene-5′-carbaldehyde (57)	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	278	C13H10O3S2	Qinghe County, Xinjiang, China	[27]
4-Hydroxy-1-(5′-methyl-[2,2′-bithiophen]-5-yl)butan-1-one (58)	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	266	C13H14O2S2	Qinghe County, Xinjiang, China	[27]
5′-(3,4-Dihydroxybut-1-yn-1-yl)-[2,2′-bithiophene]-5-carboxylic acid (59)	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	294	C13H10O4S2	Qinghe County, Xinjiang, China	[27]
4-(5′-Methyl-[2,2′-bithiophen]-5-yl)but-3-yn-1-ol (60)	Echinops ritro, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	248	C13H12OS2	Qinghe County, Xinjiang, China	[27]
Echinothiophene D (61)	Echinops grijisii, roots (Asteraceae)	EtOH/CH2Cl2-soluble fraction	298	C13H14O4S2	Zhangjiakou Hebei, China	[19]
Echinothiophene E (62)	Echinops grijisii, roots (Asteraceae)	EtOH/CH2Cl2-soluble fraction	366	C18H19ClO2S2	Zhangjiakou Hebei, China	[19]
Echinothiophene F (63)	Echinops grijisii, roots (Asteraceae)	EtOH/CH2Cl2-soluble fraction	432	C23H28O4S2	Zhangjiakou Hebei, China	[19]
2-Prop-1-inyl-5′-(2-hydroxy-3-chloropropyl) dithiophene (64)	Echinops grijisii, roots (Asteraceae)	EtOH/CH2Cl2-soluble fraction	282	C13H11ClOS2	Zhangjiakou Hebei, China	[19]
Ecliprostin A (65)	Eclipta prostrata, aerial parts (Asteraceae)	EtOH/EtOAc-soluble fraction	348	C18H20O3S2	Mount Kunyu area, Shandong, China	[18]
Ecliprostin B (66)	Eclipta prostrata, aerial parts (Asteraceae)	EtOH/EtOAc-soluble fraction	348	C18H20O3S2	Mount Kunyu area, Shandong, China,	[18]
Ecliprostin C (67)	Eclipta prostrata, aerial parts (Asteraceae)	EtOH/EtOAc-soluble fraction	678	C36H38O5S4	Mount Kunyu area, Shandong, China	[18]
Echinbithiophenedimer A (68)	Echinops latifolius, roots (Asteraceae)	EtOH/n-hexane:acetone (8:1) soluble fraction	492	C26H20O2S4	Mentougou, Beijing, China	[17]
Echinbithiophenedimer B (69)	Echinops latifolius, roots (Asteraceae)	EtOH/n-hexane:acetone (8:1) soluble fraction	492	C26H20O2S4	Mentougou, Beijing, China	[17]
Echinbithiophenedimer C (70)	Echinops latifolius, roots (Asteraceae)	EtOH/n-hexane:acetone (8:1) soluble fraction	492	C26H20O2S4	Mentougou, Beijing, China	[17]
(R)-(5′-(3,4-dihydroxybut-1-yn-1-yl)-[2,2′-bithiophen]-5-yl)methyl 3-methylisovalerate (71)	Eclipta prostrata, aerial parts (Asteraceae)	EtOH/PE-soluble fraction	364	C18H20O4S2	Mount Kunyu area, Shandong, China	[38]
5-[l-(4-hydroxybut-l-ynyl)]-2,2′-bithiophene-5′-carbaldehyde (72)	Eclipta prostrata, aerial parts (Asteraceae)	EtOH/PE-soluble fraction	262	C13H10O2S2	Mount Kunyu area, Shandong, China	[38]
5′-Hydroxymethyl-5-(3-butene-1-ynyl)-2,2′-bithiophene (73)	Eclipta prostrata, aerial parts (Asteraceae)	EtOH/PE-soluble fraction	246	C13H10OS2	Mount Kunyu area, Shandong, China	[38]
4-(5′-(hydroxymethyl)-[2,2′-bithiophene]-5-yl)but-3-yn-1-ol) (Thio1) (74)	Tagetes patula, aerial parts (Asteraceae)	Synthesis	264	C13H12O2S2	-	[46]
III. Terthiophene
2,2′:5′,2′′-Terthiophene (α-Terthienyl) (α-T) (75)	Echinops grijisii, roots (Asteraceae)	MeOH/EtOAc-soluble fraction	248	C12H8S3	Nantou, Taiwan	[28]
	Eclipta prostrata, aerial parts (Asteraceae)	MeOH/EtOAc-soluble fraction	-	-	Seoul, Korea	[39]
	Porophyllum obscurum, aerial parts (Asteraceae)	n-Hexane extract	-	-	Las Chacras, province of San Luis, Argentina	[40]
	Echinops grijisii, roots (Asteraceae)	Essential oil, hydrodistillation	-	-	Nanjing, Jiangsu, China	[41]
	Echinops albicaulis, aerial parts (Asteraceae)	MeOH/CH2Cl2 soluble fraction	-	-	Malaysary gorge, Kazakhstan	[42]
5-Formyl-2,2′:5′,2′′-terthiophene (Ecliptal) (76)	Eclipta prostrata, aerial parts (Asteraceae)	MeOH/EtOAc-soluble fraction	276	C13H8OS3	Seoul, Korea	[39]
	Eclipta prostrata, whole plant (Asteraceae)	EtOH/n-hexane-soluble fraction	-	-	Seoul, Korea	[47]
	Tagetes minuta, aerial parts (Asteraceae)	MeOH/n-hexane-soluble fraction	-	-	Al-Baha city, Saudi Arabia	[43]
5-Hydroxymethyl-2,2′:5′,2′′-terthiophene (α-Terthienylmethanol) (77)	Eclipta prostrata, aerial parts (Asteraceae)	MeOH/EtOAc-soluble fraction	278	C13H10OS3	Seoul, Korea	[39]
	Eclipta prostrata, whole plant (Asteraceae)	EtOH/n-hexane-soluble fraction	-	-	Seoul, Korea	[47]
	Eclipta prostrata, whole plant (Asteraceae)	EtOH/n-hexane-soluble fraction	-	-	Seoul, Korea	[20]
3′-Methoxy-2,2′:5′,2′′-terthiophene (78)	Eclipta prostrata, aerial parts (Asteraceae)	MeOH/EtOAc-soluble fraction	278	C13H10OS3	Seoul, Korea	[39]
5-Hydroxymethyl-(2,2′:5′,2′′)-terthienyl angelate (79)	Eclipta prostrata, whole plant (Asteraceae)	EtOH/n-hexane-soluble fraction	360	C18H16O2S3	Seoul, Korea	[47]
5-Hydroxymethyl-(2,2′:5′,2′′)-terthienyl tiglate (80)	Eclipta prostrata, whole plant (Asteraceae)	EtOH/n-hexane-soluble fraction	360	C18H16O2S3	Seoul, Korea	[47]
5-Methoxy-(2,2′:5′,2′′)-terthiophene (81)	Eclipta prostrata, whole plant (Asteraceae)	EtOH/n-hexane-soluble fraction	292	C14H12OS3	Seoul, Korea	[47]
3′-Hydroxy-2,2′:5′,2′′-terthiophene-3′-O-β-D-glucopyranoside (82)	Eclipta prostrata, aerial parts (Asteraceae)	MeOH/EtOAc-soluble fraction	426	C18H18O6S3	Seoul, Korea	[39]
IV. Quinquethiophenes
Thiotagetin A (83)	Tagetes minuta, aerial parts, (Asteraceae)	MeOH/n-hexane-soluble fraction	540	C27H24O2S5	Al-Baha city, Saudi Arabia	[48]
V. Miscellaneous
Sibiricumthionol (84)	Xanthium sibiricum, fruits (Asteraceae)	MeOH/CH2Cl2-soluble fraction	197	C9H11NO2S	Helen town, Heilongjiang, China	[49]
(+)-Xanthienopyran (85)	Xanthium sibiricum, fruits (Asteraceae)	MeOH/CH2Cl2-soluble fraction	316	C17H16O4S	Helen town, Heilongjiang, China	[49]
Rupestriene A (86)	Artemisia rupestris, whole plant (Asteraceae)	EtOH/CH3Cl-soluble fraction	280	C15H20O3S	Xinjiang Uygur Autonomous,China	[36]
7-[1-(Thiophene-5-yl)-1-formamido]-3-propylenyl-3-cephem-4-carboxylic acid (CAx1) (87)	Endolithic Streptomyces sp. AL51	Spore MeOH extract	350	C15H14N2O4S2	Granite rock, Mylliem, Meghalaya, India	[50]
2,5-Bis(5-tert-butyl-2-benzoxazolyl)thiophene (88)	Marine-derived actinomycete Streptomyces sp. G278 isolated from echinoderm Holothuria edulis	Fermentation broth	430	C26H26N2O2S	Cu Lao Cham- Quang Nam, Vietnam	[51]
Thiocarboxylic A (89)	Penicillium sp. sb62 was isolated from soil collected near the fibrous roots ofSchisandra bicolor var. tuberculata (Schisandraceae)	EtOAc of fermented material	332	C18H20O4S	Xinning, Hunan, China	[16]
Thiocarboxylic B (90)	Penicillium sp. sb62 was isolated from soil collected near the fibrous roots ofSchisandra bicolor var. tuberculata (Schisandraceae)	EtOAc of fermented material	376	C19H20O6S	Xinning, Hunan, China	[16]
Thiocarboxylic C1 (91)	Penicillium sp. sb62 was isolated from soil collected near the fibrous roots ofSchisandra bicolor var. tuberculata (Schisandraceae)	EtOAc of fermented material	348	C18H20O5S	Xinning, Hunan, China	[16]
Thiocarboxylic C2 (92)	Penicillium sp. sb62 was isolated from soil collected near the fibrous roots ofSchisandra bicolor var. tuberculata (Schisandraceae)	EtOAc of fermented material	348	C18H20O5S	Xinning, Hunan, China	[16]
Thiocarboxylic D1 (93)	Penicillium sp. sb62 was isolated from soil collected near the fibrous roots of Schisandra bicolor var. tuberculata (Schisandraceae)	EtOAc of fermented material	350	C18H22O5S	Xinning, Hunan, China	[16]
Thiocarboxylic D2 (94)	Penicillium sp. sb62 was isolated from soil collected near the fibrous roots ofSchisandra bicolor var. tuberculata (Schisandraceae)	EtOAc of fermented material	350	C18H22O5S	Xinning, Hunan, China	[16]
Rupestriene D (95)	Artemisia rupestris, whole plant (Asteraceae)	EtOH/PE-soluble fraction	234	C13H14O2S	Xinjiang Uygur Autonomous,China	[15]
Rupestriene E (96)	Artemisia rupestris, whole plant (Asteraceae)	EtOH/EtOAc-soluble fraction	294	C15H18O4S	Xinjiang Uygur Autonomous,China	[15]

Scheme 1

Proposed biosynthetic pathway of dimeric bithiophenes 68–70 from arctinol-b (49) [17].

Scheme 2

Proposed biosynthetic pathway of 46 [44,52,53,54].

Table 2

Biological activities of naturally occurring thiophenes.

Compound Name	Biological Activity	Assay, Organism, or Cell Line	Biological Results		Ref.
			Compound	Positive Control
Foetithiophene F (6)	Antimicrobial	Broth microdilution/B. cereus	50 µg/mL (MIC)	Gentamicin 10 µg/mL (MIC)	[25]
5-Propinyl-thiophene-2-carboxylic acid (7)	In vitro anti-inflammatory/NO	LPS-stimulated production in BV-2 microglial cells	79.5 µM (IC50)	Quercetin 16.3 µM (IC50)	[26]
3-Hydroxy-5-propinyl-2-acetyl-thiophene (8)	In vitro anti-inflammatory/NO	LPS-stimulated production in BV-2 microglial cells	98.5 µM (IC50)	Quercetin 16.3 µM (IC50)	[26]
2-(3,4-Dihydroxybut-1-ynyl)-5-(penta-1,3-diynyl)thiophene (9)	In vitro anti-inflammatory/NO	LPS-stimulated production in the RAW 264.7 cell line	2.5 µg/mL (IC50)	Indomethacin 65.4 µg/mL (IC50)	[28]
	Cytotoxicity	Resazurin reduction/CEM/ADR5000	21.09 µM (IC50)	Doxorubicin 195.12 µM (IC50)	[30]
	Cytotoxicity	Resazurin reduction/CCRF-CEM	46.96 µM (IC50)	Doxorubicin 0.20 µM (IC50)	[30]
	Antimicrobial	INT/E. coli	64.0 µg/mL (MIC)	Chloramphenicol 64.0 µg/mL (MIC)	[29]
	Antimicrobial	INT/E. aerogenes	64.0 µg/mL (MIC)	Chloramphenicol 16.0 µg/mL (MIC)	[29]
	Antimicrobial	INT/K. pneumoniae	64.0 µg/mL (MIC)	Chloramphenicol 16.0 µg/mL (MIC)	[29]
	Antimicrobial	INT/P. stuartil	64.0 µg/mL (MIC)	Chloramphenicol 128.0 µg/mL (MIC)	[29]
	Antimicrobial	INT/E. cloacae	256.0 µg/mL (MIC)	Chloramphenicol 256.0 µg/mL (MIC)	[29]
	Antimicrobial	INT/P. aeruginosa	256.0 µg/mL (MIC)	Chloramphenicol 16.0 µg/mL (MIC)	[29]
2-(Penta-1,3-diyn-1-yl)-5–(4-acetoxy-3-hydroxybuta-1-yn-1-yl) thiophene (11)	CYP2A6 inhibition	Enzymatic reconstitution	6.43 µM (IC50)	Methoxsalen 0.19 µM (IC50)	[31]
	CYP2A13inhibition	Enzymatic reconstitution	6.18 µM (IC50)	Methoxsalen 0.43 µM (IC50)	[31]
Echinothiophene A (15)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	0.42 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			1.44 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/Fusarium solani	64.0 µg/mL (MIC)	Carbendazim 0.5 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. vasinfectum	16.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. niveum	8.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Phytophthora infestans	128.0 µg/mL (MIC)	Carbendazim 256.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	16.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternata	4.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
Echinothiophene B (16)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	2.65 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			9.23 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/Fusarium solani	32.0 µg/mL (MIC)	Carbendazim 0.5 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. vasinfectum	64.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. niveum	16.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Phytophthora infestans	256.0 µg/mL (MIC)	Carbendazim 256.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	8.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternataalternata	8.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
Echinothiophene C (17)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	16.55 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			18.17 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/F. oxysporum f. sp. vasinfectum	128.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. niveum	256.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	128.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternataalternata	32.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
2-(Pro-1-ynyl)-5-(5,6-dihydroxypenta-1,3-diynyl) thiophene (PYDDT) (18)	CYP2A6 inhibition	Enzymatic reconstitution	3.90 µM (IC50)	Methoxsalen 0.19 µM (IC50)	[31]
	CYP2A13 inhibition	Enzymatic reconstitution	2.40 µM (IC50)	Methoxsalen 0.43 µM (IC50)	[31]
5-(1,2-Dihydroxyethyl)-2-(E)-hept-5-ene-1,3-diynylthiophene (19)	In vitro anti-inflammatory/NO	LPS-stimulated production in the RAW 264.7 cell line	28.2 µM (IC50)	-Indomethacin 13.2 µM (IC50)-Aminoguanidine 24.2 µM (IC50)	[34]
5-(1,2-Dihydroxy-ethyl)-2-(Z)-hept-5-ene-1,3-diynylthiophene (20)	In vitro anti-inflammatory/NO	LPS-stimulated production in the RAW 264.7 cell line	12.8 µM (IC50)	-Indomethacin 13.2 µM (IC50)-Aminoguanidine 24.2 µM (IC50)	[34]
2-(Prop-1-inyl)-5-(6-acetoxy-5-hydroxyhexa-1,3-diinyl) thiophene (22)	CYP2A6 inhibition	Enzymatic reconstitution	4.44 µM (IC50)	Methoxsalen 0.19 µM (IC50)	[31]
	CYP2A13 inhibition	Enzymatic reconstitution	2.94 µM (IC50)	Methoxsalen 0.43 µM (IC50)	[31]
3′′R-Pluthiophenol (23)	In vitro anti-inflammatory/NO	LPS-stimulated production in RAW 264.7 macrophages cells	84.5 (NRC % inhibition)	Dexamethasone 62.2 (NRC % inhibition)	[35]
3′′R-Pluthiophenol-4′′-acetate (24)	In vitro anti-inflammatory/NO	LPS-stimulated production in RAW 264.7 macrophages cells	83.4 (NRC % inhibition)	Dexamethasone 62.2 (NRC % inhibition)	[35]
3′′-Ethoxy-3′′S-pluthiophenol (25)	In vitro anti-inflammatory/NO	LPS-stimulated production in RAW 264.7 macrophages cells	86.9 (NRC % inhibition)	Dexamethasone 62.2 (NRC % inhibition)	[35]
3′′-Ethoxy-3′′S-pluthiophenol-4′′-acetate (26)	In vitro anti-inflammatory/NO	LPS-stimulated production in RAW 264.7 macrophages cells	90.1 (NRC % inhibition)	Dexamethasone 62.2 (NRC % inhibition)	[35]
Rupestriene B (27)	In vitro anti-inflammatory/NO	LPS-stimulated production in BV-2 microglial cells	8.5 µM (IC50)	Quercetin 4.3 µM (IC50)	[36]
Rupestriene C (28)	In vitro anti-inflammatory/NO	LPS-stimulated production in BV-2 microglial cells	5.3 µM (IC50)	Quercetin 4.3 µM (IC50)	[36]
5-(3,4-Dihydroxybut-1-ynyl)-2,2′-bithiophene (33)	In vitro anti-inflammatory/NO	LPS-stimulated production in the RAW 264.7 cell line	20.0 µg/mL (IC50)	Indomethacin 65.4 µg/mL (IC50)	[28]
5-(But-3-en-1-ynyl)-2,2′-bithiophene (5-BBT) (37)	Fungicidal	Broth microdilution/C. albicans ATCC 10231	7.81 µg/mL (MFC) in light	Amphotericin B 0.50 µg/mL (MFC)Fluconazole ˃ 64 µg/mL (MFC)Itraconazole ˃ 16 µg/mL (MFC)	[40]
	Larvicidal	Larval mortality/Aedes albopictus	0.34 µg/mL (LC50)	Rotenone 3.75 µg/mL (LC50)	[41]
	Larvicidal	Larval mortality/Aedes albopictus	0.72 µg/mL (LC95)	Rotenone 9.45 µg/mL (LC95)	[41]
	Larvicidal	Larval mortality/Anopheles sinensis	1.36 µg/mL (LC50)	Rotenone 1.25 µg/mL (LC50)	[41]
	Larvicidal	Larval mortality/Anopheles sinensis	1.93 µg/mL (LC95)	Rotenone 2.24 µg/mL (LC95)	[41]
	Larvicidal	Larval mortality/Culex pipiens pallens	0.12 µg/mL (LC50)	Rotenone 1.88 µg/mL (LC50)	[41]
	Fungicidal	Larval mortality/Culex pipiens pallens	0.18 µg/mL (LC95)	Rotenone 3.74 µg/mL (LC95)	[41]
	Fungicidal	Broth microdilution/C. albicans ATCC 10231	62.50 µg/mL (MFC) in low oxygen and light	-	[59]
	Fungicidal	Broth microdilution/C. albicans ATCC 10231	7.81 µg/mL (MFC) in normal oxygen and light	-	[59]
5-(4-Isovaleroyloxybut-1-ynyl)-2,2′-bithiophene (5-IBT) (38)	Larvicidal	Larval mortality/Aedes albopictus	0.45 µg/mL (LC50)	Rotenone 3.75 µg/mL (LC50)	[41]
	Larvicidal		0.66 µg/mL (LC95)	Rotenone 9.45 µg/mL (LC95)	[41]
	Larvicidal	Larval mortality/Anopheles sinensis	5.36 µg/mL (LC50)	Rotenone 1.25 µg/mL (LC50)	[41]
	Larvicidal		11.26 µg/mL (LC95)	Rotenone 2.24 µg/mL (LC95)	[41]
	Larvicidal	Larval mortality/Culex pipiens pallens	0.33 µg/mL (LC50)	Rotenone 1.88 µg/mL (LC50)	[41]
	Larvicidal		0.54 µg/mL (LC95)	Rotenone 3.74 µg/mL (LC95)	[41]
5-(4-Hydroxy-1-butynyl)-2,2′-bithiophene (43)	In vitro anti-inflammatory/NO	LPS-stimulated production in the RAW 264.7 cell line	6.7 µg/mL (IC50)	Indomethacin 65.4 µg/mL (IC50)	[28]
	Fungicidal	Broth microdilution/C. albicans ATCC 10231	3.90 µg/mL (MFC) in light	-Amphotericin B 0.50 µg/mL (MFC)-Fluconazole ˃ 64 µg/mL (MFC)-Itraconazole ˃ 16 µg/mL (MFC)	[40]
	Antimicrobial	Broth microdilution/S. aureus ATCC 2592	8.0 µg/mL (MIC)	Levofloxacin 8.0 μg/mL (MIC)	[27]
	Antimicrobial	Broth microdilution/E. coli ATCC 25922	64.0 µg/mL (MIC)	Levofloxacin 16.0 μg/mL (MIC)	[27]
	Antimicrobial	Broth microdilution/C. albicans ATCC2002	64.0 µg/mL (MIC)	Levofloxacin 64.0 μg/mL (MIC)	[27]
	Fungicidal	Broth microdilution/C. albicans ATCC 10231	250.0 µg/mL (MFC) in low oxygen and light	-	[59]
	Fungicidal	Broth microdilution/C. albicans ATCC 10231	3.90 µg/mL (MFC) in normal oxygen and light	-	[59]
	Anti-inflammatory	Colorimetric/5-LOX	41.82 µM (IC50)	Indomethacin 0.89 µM (IC50)	[43]
5-(4-Acetoxy-1-butynl)-2,2′-bithiophene (44)	Fungicidal	Broth microdilution/C. albicans ATCC 10231	7.81 µg/mL (MFC) in light	-Amphotericin B 0.50 µg/mL (MFC)-Fluconazole ˃ 64 µg/mL (MFC)-Itraconazole ˃ 16 µg/mL (MFC)	[40]
	Fungicidal	Broth microdilution/C. albicans ATCC 10231	62.50 µg/mL (MFC) in low oxygen and light	-	[59]
	Fungicidal	Broth microdilution/C. albicans ATCC 10231	7.81 µg/mL (MFC) in normal oxygen and light	-	[59]
6-Methoxy-arctinol-b (48)	In vitro anti-inflammatory/NO	LPS-stimulated production in the RAW 264.7 cell line	30.6 µM (IC50)	-Indomethacin 13.2 µM (IC50)-Aminoguanidine 24.2 µM (IC50)	[34]
	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	5.83 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			7.05 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/Fusarium solani	128.0 µg/mL (MIC)	Carbendazim 0.5 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. vasinfectum	256.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. niveum	128.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	32.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternataalternata	32.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
Arctinol-b (49)	In vitro anti-inflammatory/NO	LPS-stimulated production in the RAW 264.7 cell line	48.7 µM (IC50)	-Indomethacin 13.2 µM (IC50)-Aminoguanidine 24.2 µM (IC50)	[34]
	Antimicrobial	Broth microdilution/S. aureus ATCC 2592	8.0 µg/mL (MIC)	Levofloxacin 8.0 μg/mL (MIC)	[27]
	Antimicrobial	Broth microdilution/E. coli ATCC 25922	64.0 µg/mL (MIC)	Levofloxacin 16.0 μg/mL (MIC)	[27]
	Antimicrobial	Broth microdilution/C. albicans ATCC2002	64.0 µg/mL (MIC)	Levofloxacin 64.0 μg/mL (MIC)	[27]
	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	13.48 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			14.72 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/F. oxysporum f. sp. vasinfectum	256.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. niveum	64.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Phytophthora infestans	128.0 µg/mL (MIC)	Carbendazim 256.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	32.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternata	64.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
Arctinone-b (50)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	1.14 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			2.00 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/F. oxysporum f. sp. vasinfectum	256.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	64.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternata	128.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
Arctinol (51)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	15.90 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			17.82 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/F. oxysporum f. sp. vasinfectum	256.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. niveum	128.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Phytophthora infestans	128.0 µg/mL (MIC)	Carbendazim 256.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	32.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternata	16.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
Arctinal (52)	Antimicrobial	Broth microdilution/S. aureus ATCC 2592	32.0 µg/mL (MIC)	Levofloxacin 8.0 μg/mL (MIC)	[19]
	Antimicrobial	Broth microdilution/E. coli ATCC 25922	64.0 µg/mL (MIC)	Levofloxacin 16.0 μg/mL (MIC)	[19]
	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	2.62 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			8.75 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/F. oxysporum f. sp. vasinfectum	64.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. niveum	128.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	32.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternata	64.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
Arctinol A (53)	Antimicrobial	Broth microdilution/S. aureus ATCC 2592	8.0 µg/mL (MIC)	Levofloxacin 8.0 μg/mL (MIC)	[27]
	Antimicrobial	Broth microdilution/E. coli ATCC 25922	64.0 µg/mL (MIC)	Levofloxacin 16.0 μg/mL (MIC)	[27]
5′-(3,4-Dihydroxybut-1-yn-1-yl)-[2,2′-bithiophene]-5-carbaldehyde (57)	Antimicrobial	Broth microdilution/S. aureus ATCC 2592	128.0 µg/mL (MIC)	Levofloxacin 8.0 μg/mL (MIC)	[27]
	Antimicrobial	Broth microdilution/E. coli ATCC 25922	256.0 µg/mL (MIC)	Levofloxacin 16.0 μg/mL (MIC)	[27]
	Antimicrobial	Broth microdilution/C. albicans ATCC2002	256.0 µg/mL (MIC)	Levofloxacin 64.0 μg/mL (MIC)	[27]
4-Hydroxy-1-(5′-methyl-[2,2′-bithiophen]-5-yl)butan-1-one (58)	Antimicrobial	Broth microdilution/S. aureus ATCC 2592	8.0 µg/mL (MIC)	Levofloxacin 8.0 μg/mL (MIC)	[27]
	Antimicrobial	Broth microdilution/E. coli ATCC 25922	32.0 µg/mL (MIC)	Levofloxacin 16.0 μg/mL (MIC)	[27]
	Antimicrobial	Broth microdilution/C. albicans ATCC2002	32.0 µg/mL (MIC)	Levofloxacin 64.0 μg/mL (MIC)	[27]
5′-(3,4-Dihydroxybut-1-yn-1-yl)-[2,2′-bithiophene]-5-carboxylic acid (59)	Antimicrobial	Broth microdilution/S. aureus ATCC 2592	256.0 µg/mL (MIC)	Levofloxacin 8.0 μg/mL (MIC)	[27]
Echinothiophene D (61)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	2.57 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			1.80 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/Fusarium solani	32.0 µg/mL (MIC)	Carbendazim 0.5 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. vasinfectum	128.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. niveum	32.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Phytophthora infestans	256.0 µg/mL (MIC)	Carbendazim 256.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	8.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternata	16.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
Echinothiophene E (62)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	8.28 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			9.12 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/Fusarium solani	64.0 µg/mL (MIC)	Carbendazim 0.5 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. vasinfectum	32.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. niveum	128.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Phytophthora infestans	256.0 µg/mL (MIC)	Carbendazim 256.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	32.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternata	16.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
Echinothiophene F (63)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	20.13 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			18.41 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/F. oxysporum f. sp. niveum	128.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	256.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternata	64.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
2-Prop-1-inyl-5′-(2-hydroxy-3-chloropropyl) dithiophene (64)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	0.91 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			0.86 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
	Antifungal	Broth microdilution/Fusarium solani	64.0 µg/mL (MIC)	Carbendazim 0.5 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. vasinfectum	32.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[19]
		Broth microdilution/F. oxysporum f. sp. niveum	4.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Phytophthora infestans	32.0 µg/mL (MIC)	Carbendazim 256.0 µg/mL (MIC)	[19]
		Broth microdilution/Colletotrichum gloeosporioides	4.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[19]
		Broth microdilution/Alternaria alternata	4.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[19]
Ecliprostin A (65)	Antibacterial	Broth microdilution/S. aureus	25.0 µM (MIC)	Penicillin 0.156 µM (MIC)	[18]
Ecliprostin B (66)	Antibacterial	Broth microdilution/S. aureus	6.25 µM (MIC)	Penicillin 0.156 µM (MIC)	[18]
Ecliprostin C (67)	Antibacterial	Broth microdilution/S. aureus	25.0 µM (MIC)	Penicillin 0.156 µM (MIC)	[18]
Echinbithiophenedimer A (68)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	16.53 µg/mL (LC50) in light	Ethoprophos 36.15 (LC50) in lightα-Terthienyl 0.62 (LC50) in light	[17]
			18.17 µg/mL (LC50) in dark	Ethoprophos 31.94 (LC50) in darkα-Terthienyl 2.23 (LC50) in dark	[17]
	Antifungal	Broth microdilution/Alternaria alternata;	16.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[17]
		Broth microdilution/Pyricularia oryzae	16.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[17]
		Broth microdilution/Fusarium oxysporum	32.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[17]
		Broth microdilution/Colletotrichum gloeosporioides	64.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[17]
		Broth microdilution/Phytophthora infestans	128.0 µg/mL (MIC)	Carbendazim 256.0 µg/mL (MIC)	[17]
Echinbithiophenedimer B (69)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	13.88 µg/mL (LC50) in light	Ethoprophos 36.15 (LC50) in lightα-Terthienyl 0.62 (LC50) in light	[17]
			16.28 µg/mL (LC50) in dark	Ethoprophos 31.94 (LC50) in darkα-Terthienyl 2.23 (LC50) in dark	[17]
	Antifungal	Broth microdilution/Alternaria alternata	16.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[17]
		Broth microdilution/Pyricularia oryzae	16.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[17]
		Broth microdilution/Fusarium oxysporum	16.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[17]
		Broth microdilution/Colletotrichum gloeosporioides	32.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[17]
		Broth microdilution/Phytophthora infestans	128.0 µg/mL (MIC)	Carbendazim 256.0 µg/mL (MIC)	[17]
Echinbithiophenedimer C (70)	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	8.73 µg/mL (LC50) in light	Ethoprophos 36.15 (LC50) in lightα-Terthienyl 0.62 (LC50) in light	[17]
			9.39 µg/mL (LC50) in dark	Ethoprophos 31.94 (LC50) in darkα-Terthienyl 2.23 (LC50) in dark	[17]
	Antifungal	Broth microdilution/Alternaria alternata;	8.0 µg/mL (MIC)	Carbendazim 16.0 µg/mL (MIC)	[17]
		Broth microdilution/Pyricularia oryzae	8.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[17]
		Broth microdilution/Fusarium oxysporum	32.0 µg/mL (MIC)	Carbendazim 8.0 µg/mL (MIC)	[17]
		Broth microdilution/Colletotrichum gloeosporioides	32.0 µg/mL (MIC)	Carbendazim 2.0 µg/mL (MIC)	[17]
		Broth microdilution/Phytophthora infestans	128.0 µg/mL (MIC)	Carbendazim 256.0 µg/mL (MIC)	[17]
4-(5′-(hydroxymethyl)-[2,2′-bithiophene]-5-yl)but-3-yn-1-ol) (Thio1) (74)	Anthelmintic	Larval development test/Haemonchus contortus	0.3243 mg/mL (EC50)	Levamisole 1.88 mg/mL (EC50)	[46]
	Anthelmintic	Fecal egg count reduction test/Haemonchus contortus	0.1731 mg/mL (EC50)	Levamisole 1.88 mg/mL (EC50)	[46]
2,2′:5′,2′′-Terthiophene (α-Terthienyl) (75)	Cytotoxicity	MTT/SKOV3	77.23 µM (IC50)	Cisplatin 11.25 µM (IC50)	[39]
	Fungicidal	Broth microdilution/C. albicans ATCC 10231	0.24 µg/mL (MFC) in light	Amphotericin B 0.50 µg/mL (MFC)Fluconazole ˃ 64 µg/mL (MFC)Itraconazole ˃ 16 µg/mL (MFC)	[40]
	Fungicidal	Broth microdilution/C. albicans ATCC 10231	7.81 µg/mL (MFC) in low oxygen and light	-	[59]
	Fungicidal	Broth microdilution/C. albicans ATCC 10231	0.24 µg/mL (MFC) in normal oxygen and light	-	[59]
	Larvicidal	Larval mortality/Aedes albopictus	1.41 µg/mL (LC50)	Rotenone 3.75 µg/mL (LC50)	[41]
	Larvicidal		2.19 µg/mL (LC95)	Rotenone 9.45 µg/mL (LC95)	[41]
	Larvicidal	Larval mortality/Anopheles sinensis	1.79 µg/mL (LC50)	Rotenone 1.25 µg/mL (LC50)	[41]
	Larvicidal		2.54 µg/mL (LC95)	Rotenone 2.24 µg/mL (LC95)	[41]
	Larvicidal	Larval mortality/Culex pipiens pallens	1.38 µg/mL (LC50)	Rotenone 1.88 µg/mL (LC50)	[41]
	Larvicidal		2.15 µg/mL (LC95)	Rotenone 3.74 µg/mL (LC95)	[41]
	Nematicidal	Nematode Mortality/J2s of Meloidogyne incognita	0.56 µg/mL (LC50) in light	Abamectin 8.73 (LC50) in light	[19]
			1.77 µg/mL (LC50) in dark	Abamectin 9.38 (LC50) in dark	[19]
5-Formyl-2,2′:5′,2′′-terthiophene (Ecliptal) (76)	Cytotoxicity	MTT/SKOV3	24.57 µM (IC50)	Cisplatin 11.25 µM (IC50)	[39]
	Cytotoxicity	MTT/Hec1A	12.00 µM (IC50)	Cisplatin 120.42 µM (IC50)	[47]
	Cytotoxicity	MTT/Ishikawa	2.20 µM (IC50)	Cisplatin 10.11 µM (IC50)	[47]
	Anti-inflammatory	Colorimetric/5-LOX	26.18 µM (IC50)	Indomethacin 0.89 µM (IC50)	[43]
	Antibacterial	Broth microdilution/S. aureus	25.0 µM (MIC)	Penicillin G 0.156 µM (MIC)	[38]
5-Hydroxymethyl-2,2′:5′,2′′-terthiophene (α-Terthienylmethanol) (77)	Cytotoxicity	MTT/SKOV3	7.73 µM (IC50)	Cisplatin 11.25 µM (IC50)	[39]
	Cytotoxicity	MTT/Hec1A	0.38 µM (IC50)	Cisplatin 120.42 µM (IC50)	[47]
	Cytotoxicity	MTT/Ishikawa	0.35 µM (IC50)	Cisplatin 10.11 µM (IC50)	[47]
	Cytotoxicity	MTT/A2780	1.18 µM (IC50)	Cisplatin 10.80 µM (IC50)	[20]
	Cytotoxicity	MTT/SKOV3	15.51 µM (IC50)	Cisplatin 43.05 µM (IC50)	[20]
	Cytotoxicity	MTT/OVCAR3	0.20 µM (IC50)	Cisplatin 35.46 µM (IC50)	[20]
	Cytotoxicity	MTT/ES2	18.82 µM (IC50)	Cisplatin 29.58 µM (IC50)	[20]
	Antibacterial	Broth microdilution/S. aureus	25.0 µM (MIC)	Penicillin G 0.156 µM (MIC)	[38]
5-Hydroxymethyl-(2,2′:5′,2′′)-terthienyl angelate (79)	Cytotoxicity	MTT/Hec1A	129.85 µM (IC50)	Cisplatin 120.42 µM (IC50)	[47]
	Cytotoxicity	MTT/Ishikawa	6.87 µM (IC50)	Cisplatin 10.11 µM (IC50)	[47]
5-Hydroxymethyl-(2,2′:5′,2′′)-terthienyl tiglate (80)	Cytotoxicity	MTT/Hec1A	2.66 µM (IC50)	Cisplatin 120.42 µM (IC50)	[47]
	Cytotoxicity	MTT/Ishikawa	9.68 µM (IC50)	Cisplatin 10.11 µM (IC50)	[47]
5-Methoxy-(2,2′:5′,2′′)-terthiophene (81)	Cytotoxicity	MTT/Hec1A	1.38 µM (IC50)	Cisplatin 120.42 µM (IC50)	[47]
	Cytotoxicity	MTT/Ishikawa	7.12 µM (IC50)	Cisplatin 10.11 µM (IC50)	[47]
3′-Hydroxy-2,2′:5′,2′′-terthiophene-3′-O-β-D-glucopyranoside (82)	Cytotoxicity	MTT/SKOV3	58.20 µM (IC50)	Cisplatin 11.25 µM (IC50)	[39]
Thiotagetin A (83)	Cytotoxicity	MTT/KB	2.03 μg/mL (ED50)	Adriamycin 0.26 μg/mL (ED50)	[48]
	Cytotoxicity	MTT/MCF-7	3.88 μg/mL (ED50)	Adriamycin 0.07 μg/mL (ED50)	[48]
Rupestriene A (86)	In vitro anti-inflammatory/NO	LPS-stimulated production in BV-2 microglial cells	20.3 µM (IC50)	Quercetin 4.3 µM (IC50)	[36]
	Neuraminidase inhibitory activity	Fluorescence-based assay	351.15 µM (IC50)	Oseltamivir acid 77.91 µM (IC50)	[15]
7-[1-(Thiophene-5-yl)-1-formamido]-3-propylenyl-3-cephem-4-carboxylic acid (CAx1) (87)	Antibacterial	Broth microdilution/S. aureus MTCC 740	0.2 µg/mL (MIC)2.0 µg/mL (MBC)	Penicillin 32.0 µg/mL (MIC)64.0 µg/mL (MBC)	[40]
	Antibacterial	Broth microdilution/B. subtilis MTCC 736	0.25 µg/mL (MIC)0.5 µg/mL (MBC)	Penicillin 0.5 µg/mL (MIC)4.0 µg/mL (MBC)	[50]
	Antibacterial	Broth microdilution/E. coli MTCC 739	4.0 µg/mL (MIC)8.0 µg/mL (MBC)	Penicillin 4.0 µg/mL (MIC)16.0 µg/mL (MBC)	[50]
	Antibacterial	Broth microdilution/K. pneumonia MTCC 661	4.0 µg/mL (MIC)16.0 µg/mL (MBC)	Penicillin 16.0 µg/mL (MIC)64.0 µg/mL (MBC)	[50]
2,5-Bis(5-tert-butyl-2-benzoxazolyl)thiophene (88)	Antimicrobial	Broth microdilution/E. faecalis ATCC29212	256.0 µg/mL (MIC)	Streptomycin 256.0 μg/mL (MIC)	[51]
Thiocarboxylic A (89)	Antimicrobial	Broth microdilution/E. coli ATCC35218	1.7 µg/mL (MIC)	Streptomycin 2.3 µg/mL (MIC)	[16]
		Broth microdilution/S. aureus ATCC25923	1.7 µg/mL (MIC)	Streptomycin 0.1 µg/mL (MIC)	[16]
		Broth microdilution/C. albicans ATCC10231	3.3 µg/mL (MIC)	Amphotericin B 0.1 µg/mL (MIC)	[16]
Thiocarboxylic B (90)	Antimicrobial	Broth microdilution/E. coli ATCC35218	0.9 µg/mL (MIC)	Streptomycin 2.3 µg/mL (MIC)	[16]
		Broth microdilution/S. aureus ATCC25923	1.9 µg/mL (MIC)	Streptomycin 0.1 µg/mL (MIC)	[16]
		Broth microdilution/C. albicans ATCC10231	3.9 µg/mL (MIC)	Amphotericin B 0.1 µg/mL (MIC)	[16]
Thiocarboxylic C1 (91)	Antimicrobial	Broth microdilution/E. coli ATCC35218	7.0 µg/mL (MIC)	Streptomycin 2.3 µg/mL (MIC)	[16]
		Broth microdilution/S. aureus ATCC25923	3.5 µg/mL (MIC)	Streptomycin 0.1 µg/mL (MIC)	[16]
		Broth microdilution/C. albicans ATCC10231	7.0 µg/mL (MIC)	Amphotericin B 0.1 µg/mL (MIC)	[16]
Thiocarboxylic C2 (92)	Antimicrobial	Broth microdilution/E. coli ATCC35218	7.0 µg/mL (MIC)	Streptomycin 2.3 µg/mL (MIC)	[16]
		Broth microdilution/S. aureus ATCC25923	3.5 µg/mL (MIC)	Streptomycin 0.1 µg/mL (MIC)	[16]
		Broth microdilution/C. albicans ATCC10231	7.0 µg/mL (MIC)	Amphotericin B 0.1 µg/mL (MIC)	[16]
Thiocarboxylic D1 (93)	Antimicrobial	Broth microdilution/E. coli ATCC35218	3.5 µg/mL (MIC)	Streptomycin 2.3 µg/mL (MIC)	[16]
		Broth microdilution/S. aureus ATCC25923	3.5 µg/mL (MIC)	Streptomycin 0.1 µg/mL (MIC)	[16]
		Broth microdilution/C. albicans ATCC10231	7.0 µg/mL (MIC)	Amphotericin B 0.1 µg/mL (MIC)	[16]
Thiocarboxylic D2 (94)	Antimicrobial	Broth microdilution/E. coli (ATCC35218)	3.5 µg/mL (MIC)	Streptomycin 2.3 µg/mL (MIC)	[16]
		Broth microdilution/S. aureus ATCC25923	3.5 µg/mL (MIC)	Streptomycin 0.1 µg/mL (MIC)	[16]
		Broth microdilution/C. albicans ATCC10231	7.0 µg/mL (MIC)	Amphotericin B 0.1 µg/mL (MIC)	[16]
Rupestriene D (95)	Neuraminidase inhibitory activity	Fluorescence-based assay	986.54 µM (IC50)	Oseltamivir acid 77.91 µM (IC50)	[15]
Rupestriene E (96)	Neuraminidase inhibitory activity	Fluorescence-based assay	365.40 µM (IC50)	Oseltamivir acid 77.91 µM (IC50)	[15]

Natural proteins (NP) are biologically active molecules with a myriad of structural and functional diversity. They enable the innovative design of synthetic compounds used in medicines, along with many more crucial aspects of molecular medicine, including but not limited to anti-cancer and anti-viral drugs currently in use. Many of them have proved to be incredibly useful in treating a plethora of diseases. Despite its many attributes, the speed and yields of NP-based drug discovery have significantly dropped during the golden period of 1950–1960. AI can aid in structure-dependent drug discovery by predicting the protein targets of the potential NP, thus assisting in the prediction of a compound influence on the target alongside the safety considerations. SuperPred, a prediction webserver for anatomical therapeutic chemical (ATC) code and target prediction of compounds, was used to predict the potential target for these thiophene derivatives. Based on the outcomes of the SuperPred prediction, we selected cathepsin D, one of the amplest lysosomal proteases, which is also implicated in the pathogenesis of several diseases: cancer, osteoarthritis, and possibly Alzheimer’s disease. Furthermore, in silico, including ADMET properties prediction, molecular docking for the protein ligands binding interaction, and molecular dynamics to evaluate the ligand–target interaction stability under simulated physiological conditions were also implemented.

2. Structural Characterization of Thiophenes

The structures of the reported thiophenes were elucidated by various spectral tools such as 1D (one dimensional) (1H and 13C) and 2D NMR (two-dimensional nuclear magnetic resonance spectroscopy) techniques, COSY (homonuclear correlation spectroscopy), HSQC (heteronuclear single quantum coherence), HMBC (heteronuclear multiple bond correlation), and NOESY (nuclear Overhauser effect spectroscopy) combined with other methods (UV (ultraviolet), IR (infra-red), MS (mass spectroscopy), elemental analysis). The reported spectral and physical data of the newly reported thiophenes are listed in Table S1. The relative configuration was determined by NOESY and ROESY (rotating frame Overhauser effect spectroscopy), as well as by [α]D measurement [34]. The exciton coupled circular dichroism (ECCD) analysis and electronic circular dichroism (ECD) calculations were utilized to assess the absolute configuration by comparing the theoretical and experimental CD spectra [16,17,37,49]. Additionally, the determination of the absolute configuration was carried out using Mosher’s method and analyzing chemical shift differences between (S)- and (R)-MTPA [16]. The X-ray structure crystallographic analysis of the crystalline derivatives is another tool utilized for the absolute configuration determination [49]. It was found that some compounds had no names; therefore, they are named here using the AUPAC system for nomenclature. Further, some compounds had the same molecular formulae and structures with different nomenclatures. On the other hand, some metabolites had more than one name.

3. Biosynthesis of Thiophenes

The detailed biosynthesis of thiophenes was discussed previously [8]. In this work, the recently reported biosynthetic pathways was discussed.

Wu et al. reported the biogenetic pathways of dimeric bithiophenes 68–70 (Scheme 1). These compounds had an unparalleled dimeric bithiophene skeleton containing two bithiophene units linked by uncommon cyclic diether units. It was proposed that they may be originated from arctinol-b (49). For 68 and 69, the formation of the 1,3-dioxolane ring may be obtained from an aldol condensation. Firstly, a key intermediate (I) is produced from 49 by dehydration and keto–enol tautomerism. After that, an aldol condensation among 49 and I would give 68 and 69. Additionally, an intermolecular dehydration reaction between two 49 molecules forms the 1,4-dioxane unit to give 70 [17].

Compound 46 originates from oleic acid. The latter is changed into PYE (trideca-3,5,7,9,11-pentayn-l-ene) through successive desaturation steps and shortening of the chain via crepenynic acid [52]. After that, PYE is changed into 5-BBT (5-(but-3-en-1-ynyl)-2,2′-bithiophene) via introducing a sulfur atom and ring formation that is most probably a two-step reaction [44]. Repeated elongation and desaturation of BBT yield I. Then, the double bond epoxidation produces oxirane (epoxy) intermediate II, subsequent addition of chloride by chloroperoxidase forms III, which performs additional dehydrogenation to yield 46 [53,54] (Scheme 2).

4. Biological Activities of Thiophenes

The reported thiophenes were investigated for various bioactivities. In this regard, these metabolites are associated with some types of biological actions, including antimicrobial, antiviral, anti-inflammatory, larvicidal, antioxidant, insecticidal, cytotoxic, and nematicidal effects. The results of the most active metabolites are summarized.

4.1. Anti-Inflammatory Activity

Inflammation is a host body defense mechanism that enables the body to survive during injury or infection and maintains the homeostasis of tissues in noxious conditions [55].

Endogenous NO (nitric oxide) plays a critical role in maintaining the homeostasis of varied cellular functions. NO local concentrations are highly dynamic, as independent enzymatic pathways regulate the synthesis. NO has been shown to modulate inflammation, decreasing the secretion of pro-inflammatory cytokines in human alveolar macrophages challenged with bacterial lipopolysaccharides (LPS) while not altering the basal cytokine levels. Drugs used for managing inflammatory disorders relieve these ailments, but they may have life-threatening consequences [56]. Therefore, there is great enthusiasm in developing new and safe remedies for treating inflammation from natural sources. The reported studies revealed that the anti-inflammatory potential of thiophenes could be due to inhibiting the activation of the NF-κB (nuclear factor-κB) pathway that regulates the expression of pro-inflammatory cytokines and chemokines [57].

The reported studies revealed that thiophenes prohibited TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), and 5-LOX (5-lipoxygenase), as well as NO production. Thus, their inflammatory potential could be due to the inhibition of NF-κB and NO synthase [58].

Zhou et al. reported that 7 and 8 separated from Artemisia sieversiana exhibited significant anti-neuroinflammatory potential on the LPS-caused NO production in BV-2 murine microglial cells (half-maximal inhibitory concentrations (IC50s) 79.5 and 98.5 µM, respectively), compared to quercetin (IC50 16.3 µM) [26] (Figure 3 and Figure 4).

Figure 3

Structures of monothiophenes 1–11.

Figure 4

Structures of monothiophenes 12–21.

In vitro anti-inflammatory assay, compounds 23–26 obtained from Pluchea indica aerial parts possessed significant inhibitory potential toward NO production caused by LPS in RAW 264.7 macrophages at a concentration of 40 µM with % inhibition ranging from 83.4% to 90.1% compared to dexamethasone (62.2%) [35] (Figure 5).

Figure 5

Structures of monothiophenes 22–31.

On the other side, the two new thiophene polyacetylene glycosides, atracthioenynesides A (29) and B (30) isolated from Atractylodes lancea rhizomes did not show any activity in LPS-induced NO production in BV2 cells [37].

A new bithiophene, 32, along with 16 formerly separated thiophenes, 9, 10, 33–45, and 75, were purified from Echinops grijisii roots EtOAc-soluble fraction of the MeOH extract using SiO2 CC (column chromatography) eluted with n-hexane-EtOAc gradient as well as HPLC and identified by IR, UV, NMR, and HRESIMS spectroscopy [28] (Figure 6 and Figure 7).

Figure 6

Structures of compounds 32–41.

Figure 7

Structures of bithiophenes 42–52.

These compounds were assessed for anti-inflammatory activity versus RAW 264.7 cells. Only 9, 33, and 43 (IC50s 2.5, 20.0, and 6.7 µg/mL, respectively) exhibited significant in vitro anti-inflammatory potential toward LPS-boosted NO production in RAW 264.7 cells compared to indomethacin (IC50 65.4 µg/mL) in the colorimetric assay [28]. Zhang et al. purified three new derivatives: rupestrienes A–C (86, 27, and 28), Artemisia rupestris EtOH extract by SiO2, RP-18, and Sephadex CC. Rupestrienes B and C (27 and 28) displayed significant inhibitory potential (IC50 8.5 and 5.3 μM, respectively) toward LPS-caused NO production in BV-2 microglial cells, compared to quercetin (IC50 4.3 μM), 86 was weakly active (IC50 20.3 μM) [36]. Jin et al. assessed the inhibitory potential of 19, 20, 48, 49, 51, and 55 toward NO production boosted by LPS in RAW 264.7 cells. Only 19, 20, 48, and 49 exhibited moderate inhibitory potential (IC50 12.8–48.7 µM), compared to indomethacin and aminoguanidine (IC50s 13.2 and 24.2 µM, respectively) (Table 2). On the other side, 51 and 55 did not have any activity (IC50 ˃100 µM) [34]. The structure–activity relationship revealed that the monothiophenes with two acetylene units were more potent than bithiophenes with one acetylene unit. The existence of the Δ10,11 cis double bond and 1,2-diol at C-5 enhanced the inhibitory activity [34].

Compounds 43, 46, and 76 separated from aerial parts of Tagetes minuta significantly decreased NFκB p65, TNF-α, and IL-6 compared to indomethacin in the ELISA (enzyme-linked immunosorbent assay) [44]. In 2020, Ibrahim et al. reported that 43 and 76 isolated T. minuta displayed moderate anti-inflammatory potential (IC50 41.82 and 26.18 µM, respectively) in the 5-LOX colorimetric assay in comparison to indomethacin (IC50 0.89 µM) [43].

4.2. Cytotoxic Activity

Cancer is a crucial cause of death globally, accounting for ≈10 million deaths in 2020 [48,60]. There are many available medications for treating various types of cancer. However, none of them are entirely safe and effective. Many of the reported thiophenes have been assessed for cytotoxic effectiveness toward various cancer cell lines.

Four new derivatives, foetithiophenes C-F (3–6), along with foetithiophenes A (1) and B (2), were obtained from MeOH extract of Ferula foetida roots using SiO2 CC and RP-HPLC. Unfortunately, they showed no cytotoxic capacity (IC50 ˃100 µmM) versus K562 and MCF-7 cell lines in the Alamar Blue assay [25].

Additionally, 9 had more promising cytotoxic potential (IC50 21.09 µM) than doxorubicin (IC50 195.12 µM) against CEM/ADR5000 (human T-cell lymphoblast-like cell line). However, it was weakly active toward CCRF-CEM (human leukemic cell line, IC50 46.96 µM) in the resazurin reduction cytotoxic assay [30].

Compounds 11, 18, and 22 isolated from Pluchea indica aerial parts were assayed for inhibitory potential on coumarin 7-hydroxylation induced by CYP2A6 (cytochrome P450 2A6) and CYP2A13 (cytochrome P450 2A13) enzymes, using enzymatic reconstitution assay [31]. The human liver cytochrome P450 (CYP) 2A13 and 2A6 enzymes had a crucial function in nicotine metabolism and the activation of tobacco-specific nitrosamine carcinogens. Their prohibition could represent a strategy for smoking abstinence and decreasing risks of lung cancer and respiratory complaints. It was found that 18, 11, and 22 irreversibly prohibited CYP2A6- and CYP2A13-induced coumarin 7-hydroxylation (IC50 values 3.90 and 2.40 µM, respectively, for 18; IC50 6.43 and 6.18 µM, respectively for 11, and IC50 4.44 and 2.94 µM, respectively for 22). These metabolites could aid in smoking stoppage and lessened risks of lung cancer and respiratory illnesses [31].

Xu et al. reported that the treatment of SW620 (human colon cancer) cells with PYDDT (2-(pro-1-ynyl)-5-(5,6-dihydroxypenta-1,3-diynyl) thiophene) (18) led to the induction of mitochondrial-mediated apoptosis that was featured by cleavage of PARP (poly ADP ribose polymerase), activating caspase-3 and 9, the release of cytochrome c from mitochondria, mitochondrial membrane potential loss, Bcl-2 (B-cell lymphoma 2) downregulation, and Bax mitochondrial translocation. A mechanism study revealed that PYDDT induced SW620 apoptosis through a JNK (c-Jun N-terminal kinase)/ROS (reactive oxygen species)-mediated mitochondrial pathway [33].

Ecliprostins A–C (65–67) new thiophene derivatives were separated from Eclipta prostrata. In contrast, ecliprostins A (65) and B (66) featured a bithiophenyl acetylenic skeleton, incorporating an isovalerate unit, whereas ecliprostin C (67) was a dimer of 65. They exerted no noticeable cytotoxicity versus Hela and MDA-MB-231 cell lines (Conc. 30 µM) [18].

Compounds 33, 75–78, and 82 were purified from the EtOH extract of Eclipta prostrata aerial parts by SiO2 CC (silica gel column chromatography) and purified using a reversed-phase CC. In the MTT assay, exhibited the most potent cytotoxicity on SKOV3 cells (IC50 7.73 µM) than cisplatin (IC50 11.25 µM). The terthiopenes 75, 76, and 82 showed significant cytotoxicity (IC50 values ranging from 24.57 to 77.23 µM). However, 33 and 78 were ineffective (IC50 values ˃ 100 µM) [39].

Additionally, Preya et al. reported that isolated Eclipta prostrata was a more potent cell growth inhibitor (IC50s 0.20–18.82 µM) than cisplatin (IC50 10.80 to 43.05 µM) toward a panel of human ovarian cancer cell lines; OVCAR3, SKOV3, A2780, and ES2 in the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. It caused changes in S phase-linked proteins (cyclins A and D2 and cyclin-dependent kinase 2) and induced an intracellular increase in ROS that increased the levels of p-H2AX (H2A histone family member X), resulting in DNA (deoxyribonucleic acid) damage [14,20]. A mechanism study indicated that 77 caused S-phase cell cycle arrest by inducing ROS stress and DNA damage. Therefore, 77 could be a potential therapeutic lead for treating ovarian cancer.

Sibiricumthionol (84) and (+)-xanthienopyran (85) were purified from Xanthium sibiricum fruits extract using SiO2, RP-18 (reversed phase-18), and HPLC (high-performance liquid chromatography) that were characterized by spectroscopic, X-ray, and ECCD analyses, as well as ECD calculations. These metabolites were inactive (IC50 ˃10 µM) toward HCT-116, BGC-823, HepG2, NCI-H1650, and A2780 cell lines in the MTT assay [49].

Compounds 76, 77, and 79–81 isolated from Eclipta prostrate showed prominent cytotoxic effectiveness toward Hec1A (IC50 ranging from 0.38 to 129.85 µM) and Ishikawa (IC50 ranging from 0.35 to 9.68 µM) cells compared to cisplatin (IC50 120.4 and 10.11 µM, respectively). Notably, 77 had a potent effect on Ishikawa and Hec1A cells (IC50 0.35 and 0.38 µM, respectively) [37,47]. The inhibitory effect of 77 was mediated by the induction of apoptosis, triggering caspase activation and cytochrome c release into the cytosol. Additionally, it increased the ROS intracellular level and decreased GSH (glutathione). Therefore, its apoptotic effect was attributed to the generation of reactive oxygen species via NADPH (nicotinamide adenine dinucleotide phosphate) oxidase in human endometrial cancer cells [47].

Thiotagetin A (83) purified from Tagetes minuta possessed cytotoxic capacity versus MCF-7 and KB (ED50s 3.88 and 2.03 μg/mL, respectively), compared to adriamycin (0.07 and 0.26 μg/mL, respectively) in the MTT assay [48].

4.3. Antimicrobial Activity

Infectious diseases continue to be a serious worldwide health concern. Multidrug-resistant (MDR) pathogens significantly increased morbidity and mortality rates [61]. The continuous emergence of MDR pathogens drastically reduced the efficacy of the utilized antibiotics resulting in a growth rate of therapeutic failure [62]. Accordingly, new and effective antimicrobial agents to tackle microbial infections are needed [50].

Chitsazian-Yazdi et al. assayed the antimicrobial activity of 1–6 in broth microdilution method toward B. cereus PTCC-1247, C. albicans ATCC-10231, and E. coli ATCC-8739. Whereas only 6 displayed the most potent potential (MIC 50 µg/mL) against B. cereus, compared to gentamicin (MIC 10 µg/mL) [25].

Mbaveng et al. purified 9 from the CH2Cl2 fraction of Echinops giganteus roots. It showed moderate and selective activities against E. coli ATCC-8739, E. aerogenes ATCC-13048 and -EA27, K. pneumonia ATCC11296, P. stuartii ATCC29916, E. cloacae BM47, and P. aeruginosa PA01 (MIC <100 µg/mL) in the rapid INT (p-iodonitrotetrazolium) chloride assay [29].

In 2017, Postigo et al. reported the separation and structural elucidation of 37, 43, 44, and 75 the from n-hexane extract of Porophyllum obscurum by preparative CTL (centrifugal thin layer) and TL (thin-layer) chromatography that were assayed for their fungicidal potential against C. albicans ATCC-10231 and 25 clinical strains of Candida spp. isolates as causative agents of oropharyngeal candidiasis using broth microdilution. They exhibited fungicidal effectiveness with minimum fungicidal concentrations (MFC) ranging from 0.24 to 7.81 μg/mL under UV-A irradiation, whereas 32 with (MFC 0.24 μg/mL) and 43 with (MFC 3.90 μg/mL) were the most active metabolites [40]. In 2019, Postigo et al. evaluated their photoinactivation towards C. albicans in parallel under darkness and light conditions. The results revealed that these thiophenes exhibited the highest potential under normal-light/oxygen atmosphere (MFCs ranged from 0.24 to 7.81 μg/mL). However, their effects decreased >200 times (MFCs ranged from 7.81 to 250 μg/mL) with low-oxygen conditions. On the other hand, all tested thiophenes had no antifungal potential in darkness under both oxygen conditions (MFC > 250 μg/mL). It was found that 75 was the most active photosensitizer and was the only one that generated a single oxygen at MFC. Furthermore, it did not elevate sensitivities to oxidative and osmotic stressors and did not produce leakage or apoptosis [59]. Therefore, their antifungal mechanism was proposed to be photodynamic, considering that the absence of oxygen had a passive effect on the antifungal photosensitivity capacity. Therefore, these features could encourage further assessments to confirm their potential application as photosensitizers in photodynamic antimicrobial therapy toward fungal infections [59].

Li et al. performed a broth microdilution assay for evaluating the antimicrobial potential of 7, 9, 33, 34, 43, 45, 47, 49, 52–54, and 57–60 (Figure 8) isolated from E. ritro versus E. coli, S. aureus, and C. albicans. Compounds 43, 49, 53, and 58 exhibited the same antibacterial activity toward S. aureus as levofloxacin (MIC (minimum inhibitory concentration) 8 µg/mL). Additionally, 43, 49, 52, 53, and 58 possessed activity against E. coli (MIC values of 32–64 µg/mL). On the other side, 43, 49, and 58 displayed antifungal potential toward C. albicans (MIC values of 32–64 µg/mL) that was similar or two-fold more active than levofloxacin (MIC 64 µg/mL) [27].

Figure 8

Structures of bithiophenes 53–63.

Liu et al. reported that 15, 16, 48, 51, 61, 62, and 64 possessed equivalent or better antifungal capacities toward Fusarium solani, Colletotrichum gloeosporioides, F. oxysporum f. sp. vasinfectum, Phytophthora infestans, Alternaria alternata, and F. oxysporum f. sp. niveum compared to carbendazim, whereas 17, 48, 50, 52, and 63 had weak antifungal potential (MICs from 32 to >256 µg/mL). It is noteworthy that 15 (MICs 4 and 8 µg/mL, respectively) had elevated inhibitory capacity toward A. alternata and F. oxysporum f. sp. niveum compared to 16, 17, and 62 (MICs from 8 to >256 µg/mL), indicating that acylation weakened the activity. Further, the effect of 15 and 16 versus all fungi was more than that of 17, suggesting that chlorine could enhance activity [19].

Compounds 65–67 showed moderate growth inhibition against S. aureus (MICs 25.0, 6.25, and 25.0 µM, respectively) in the broth microdilution assay, compared to penicillin (MIC 0.156 µM) [18], whilst they did not have significant activity toward Vibrio vulnificus and E. coli [18].

Echinbithiophenedimers A–C (68–70) novel dimeric bithiophenes, besides 37 and 49, were separated from Echinops latifolius using SiO2, Sephadex CC, and PTLC (Figure 9). Their antifungal potential against soil-borne fungi; Pyricularia oryzae, Alternaria alternata, Colletotrichum gloeosporioides, Fusarium oxysporum, and Phytophthora infestans were assessed in light and dark by the micro-broth dilution method. Compounds 68–70 had significant antifungal capacities toward P. oryzae and A. alternata (MICs 8–16 µg/mL), whereas 70 (MIC 8 µg/mL) displayed better antifungal potential toward A. alternata than carbendazim (MIC 16 µg/mL). Additionally, they revealed more antifungal potential (MIC 28 µg/mL) against P. infestans than carbendazim (MIC 256 µg/mL). It was found that an increased thiophene rings′ number bettered the activity [17].

Figure 9

Structures of bithiophenes 64–72.

Yu et al. purified two new thiophenes derivatives, 31 and 71, together with 9, 33, 48, 49, 71–73, 77, and 82 from Eclipta prostrata by SiO2, Sephadex CC, and RP-HPLC [38]. Only 77 and 82 exerted mild antibacterial potential toward S. aureus (MIC 25 µM) in the broth microdilution method, compared to penicillin G (MIC 0.156 µM) (Figure 10) [38].

Figure 10

Structures of bithiophenes (73 and 74), terthiophenes (75–82), and quinquethiophene 83.

Compound 87 was biosynthesized using endolithic Streptomyces sp. AL51. This compound had remarkable antibacterial potential versus both Gram-positive and -negative bacteria in the microplate broth-dilution method. It displayed higher activity than penicillin against Gram-positive S. aureus, B. subtilis, E. coli, and Klebsiella pneumonia with MIC/MBC (minimum bactericidal concentration) 0.2/2.0, 0.25/0.5, 4.0/8.0, and 4.0/16.0 µg/mL, respectively, compared to penicillin (MIC/MBC 32.0/64.0, 0.5/4.0, 4.0/16.0, and 16.0/64.0 µg/mL, respectively) [50].

Cao et al. purified 88 from the culture broth of the marine-derived actinomycete Streptomyces sp. G278 selectively prohibited Enterococcus faecalis equal to streptomycin (MIC 256 μg/mL) [51].

Six novel thiophene-furan-carboxylic acids, 89–94, were isolated from the soil-derived fungus Penicillium sp. Sb62, representing the first class of natural furan-carboxylic acids having a thiophene moiety (Figure 11). They possessed antimicrobial capacities versus E. coli, S. aureus, and C. albicans with MICs 0.9–7.0, 1.7–3.5, and 3.3–7.0 μg/mL, respectively, in the broth microdilution assay. It was observed that the absence of methoxy or a hydroxy substituent on the side chain enhanced the activity similar to 89 and 90, and the configurations of the methoxy or hydroxy groups on the side chain had a little effect as in 91, 92, 93, and 94 [16].

Figure 11

Structures of miscellaneous thiophenes 84–96.

4.4. Antimalarial Activity

Malaria represents a significant parasitic disease worldwide, which is accountable for the death of at least half a million people yearly [63]. Globally, the estimated malaria cases in 2020 are 241 million in 85 malaria-endemic countries [64]. There is currently a vast augmentation of resistance to the available antimalarial drugs, which necessitates the search to pinpoint new drugs to combat malaria [65].

Bitew et al. evaluated the antimalarial activity of 9 and 14 isolated from CH2Cl2 fraction of Echinops hoehnelii roots utilizing the standard suppressive method in Plasmodium berghei-affected mice. Compounds 9 and 14 at 50 and 100 mg/kg concentrations decreased parasitemia levels by 43.2% and 50.2% and 18.8% and 32.7%, respectively, compared to chloroquine. It was suggested that the ester functional group produced a two-fold decrease in the activity as in 14 [32].

4.5. Larvicidal Activity

Currently used larvicides are synthetic pesticides with high toxic effects on humans and other non-targeted organisms. Several reports revealed that thiophenes demonstrated toxic effect toward insects, especially larval mosquitoes. It was proposed that thiophenes showed the promising possibility to be set as natural larvicides for controlling mosquitoes.

Zhao et al. reported that E. grijsii essential oil exhibited larvicidal potential versus the fourth instar larvae of Anopheles sinensis, Culex pipiens pallens, and Aedes albopictus (LC50s (lethal concentrations 50%) s 3.43, 1.47, and 2.65 µg/mL, respectively) in the larval mortality bioassay compared to rotenone. Further, the purified metabolites; 5-BBT (5-(but-3-en-1-ynyl)-2,2′-bithiophene) (37), 5-IBT (5-(4-isovaleroyloxybut-1-ynyl)-2,2′-bithiophene) (38), and α-T (α-terthienyl) (75) possessed remarkable larvicidal effectiveness (LC50 0.34, 0.45, and 1.41 µg/mL, respectively for Ae. albopictus, LC50 1.36, 5.36, and 1.79 µg/mL, respectively for An. sinensis, and LC50 0.12, 0.33, and 1.38 µg/mL, respectively for C. pipiens pallens) compared to rotenone (LC50 3.75, 1.25, and 1.88 µg/mL, respectively) [41].

4.6. Nematicidal Activity

Nematodes and plant pathogenic fungi cause diseases that can lessen the yield and quality of several crops [66]. Chemical control utilizing synthetic-produced pesticides is a commonly used way to manage these diseases. The possible imperilment of synthetic chemicals toward non-target organisms and pesticide resistance rationalized the development of eco-friendly and safe pesticides [67]. Discovering efficient and less toxic natural pesticides has given rise to a top preference in the contemporaneous pesticide industry [68].

Compounds 15, 16, 48, 50, 52, 61, 62, and 64 showed more potent nematicidal effect toward J2s (second-stage juveniles) of Meloidogyne incognita (LC50 values ranging from 0.42 to 8.28 μg/mL in light and from 0.86 to 9.23 μg/mL in dark) than abamectin (LC50 values 9.38 μg/mL in dark and 8.73 μg/mL in light). Noticeably, 61 and 64 possessed better dark potential compared to their light potential than control. Particularly, 64 was the most powerful metabolite against J2s (LC50 values 0.91 and 0.86 μg/mL, under light and dark, respectively) [13]. Compounds 48, 49, 51, and 61–64 were regarded as non-phototoxic metabolites. It was found that the thiophene unit was fundamental for the activity. However, an increase in the number of acetylenes and chlorine enhanced the effect [13,19]. Compounds 68–70 were evaluated for their nematicidal potential toward the J2s of Meloidogyne incognita under dark and light conditions in nematode mortality bioassays. They showed potent nematicidal potential (LC50 9.39–18.17 µg/mL/dark and 8.73–16.53 µg/mL/light) compared to ethoprophos (LC50 31.94 µg/mL/dark and 36.15 µg/mL/light). However, they had weaker nematicidal influences than α-terthienyl (phototoxic thiophene), suggesting that they were non-phototoxic. Furthermore, 70 exhibited more powerful activity (LC50 8.73 and 9.39 µg/mL under light and dark, respectively) than its monomeric bithiophene 49, revealing that the dimeric bithiophene framework with a 1,4-dioxane moiety in 70 enhanced the nematicidal potential [17].

Compound 74 previously reported from Tagetes patula aerial parts was synthesized by Politi et al. It had a marked in vitro anthelmintic effect toward Haemonchus contortus, exhibiting 100% efficacy in the larval development and egg hatch tests with EC50 (effective concentration 50%) 0.3243 mg/mL and 0.1731 mg/mL, respectively, compared to levamisole (EC50 1.88 mg/mL) [46].

4.7. Antioxidant and Anti-Influenza Activities

Compounds 43, 46, and 76 exhibited moderate antioxidant potential with % DPPH scavenging activity ranging from 41.87 to 45.17 at 100 µM [44].

Two new thiophene derivatives, rupestriene D (95) and rupestriene E (96), along with rupestriene A (86) isolated from the whole plants of Artemisia rupestris using SiO2 CC and RP-HPLC. They exhibited neuraminidase inhibitory potential with IC50 values ranging from 351.15 to 986.54 µM in the fluorescence-based assay compared to oseltamivir acid (IC50 77.91 µM). Compounds 86 and 96 were more potent than 95, indicating that a free OH group at the C-3 side chain might enhance the activity [15].

5. AI Target-Based Prediction vs. (Virtual Screening), and MD (Molecular Dynamics) for Thiophene Derivatives

Cathepsin D is one of the most abundant lysosomal proteases. It is implicated in protein turnover and favored apoptosis in proteostasis disruption [69,70]. The disturbance in its regulation can lead to various health disorders. Its excessive levels outside the cell membrane and lysosomes result in the growth of tumors, migration, invasion, and angiogenesis [71,72]. Many of the available inhibitors have non-specific inhibitory effects that may cause serious side effects [73]. Therefore, the currently tested thiophene derivatives as cathepsin D inhibitors could provide marked diagnostic benefits and a new therapeutic approach.

In order to detect the suitable protein targets for the thiophene derivatives, ligand-based tools were utilized for in silico target prediction [74]. In the current study, SuperPred, a prediction webserver, was used for the anatomical therapeutic chemical (ATC) code and target predication of these compounds [75]. Based on the analysis of the results for all the predicted targets, cathepsin D with PDB (protein data bank) code 4OD9 was selected, which is considered a common target for most of the thiophene derivatives with high probability and model accuracy percent (Table 3). All the listed compounds were docked, using extra precision for maximum accuracy; the docking method was validated by redocking the inhibitor N-(3,4-dimethoxybenzyl)-Nalpha-{N-[(3,4-dimethoxyphenyl)acetyl]carbamimidoyl}-D-phenylalaninamide (2RZ) that co-crystallized with 4OD9, and RMSD values were found in an acceptable range. All the redocked inhibitors revealed the same binding interaction with the active site as the original pose. Further, an in silico ADMET properties prediction of the investigated compounds was carried out. Eventually, MD simulation was conducted to assess the ligand/target interaction under simulated physiological circumstances for compound 30, which showed high docking scores.

Table 3

The probability and model accuracy prediction for thiophene derivatives against Cathepsin D using SuperPred target prediction webserver.

9	Probability *	Model Accuracy **
9	96.04%	96.09%
20	93.69%	96.09%
28	82.30%	98.95%
29	85.79%	98.95%
30	82.76%	98.95%
33	87.63%	98.95%
43	82.00%	98.95%
46	94.79%	98.95%
57	89.12%	98.95%
67	91.20%	98.95%
68	77.77%	90.17%
70	88.21%	98.95%
75	79.91%	98.95%
76	87.50%	98.95%
77	85.75%	98.95%
80	90.87%	98.95%
81	90.32%	98.95%
82	88.71%	98.95%

* The input structure binds with the specific target, as determined by the respective target machine learning model; ** Since the model performances vary between different targets, additionally, the 10-fold cross-validation score of the respective logistic regression model is displayed.

5.1. In Silico ADMET Properties of Selected Ligands

The reported 96 thiophene derivatives were processed using the LigPrep of the Schrodinger suite [76]. The OPLS3 force field generated the 3D (three-dimensional) models with ionization states at 7.0 ± 0.2 pH. The QikProp module of the Schrodinger suite was utilized for predicting the ADME properties [77]. The predicted ADMET properties are summarized in Table 4. The ADMET analysis describes and determines the biological function, drug-likeness, physicochemical characters, and expected toxicity of the compounds. This is translated in terms of evaluating the usefulness of the molecules. The examined descriptors, such as drug likeness, solvent accessible surface area, dipole moment, molecular weight, hydrogen bond acceptor, and donor traits, aqueous solubility, octanol–water coefficient, number of likely metabolic reactions, brain/blood partition coefficient, human oral absorption, binding to human serum albumin, central nervous system activity, IC50 value for blockage of HERG K+ (human ether-a-go-go-related gene potassium) channels, and number of reactive functional groups were predicted for the reported thiophene derivatives. Most of the predicted values obtained for the compounds are in the recommended range, except for some highlighted parameters with yellow color.

Table 4

In silico predicted ADME properties of the thiophenes derivatives.

Title	mol MW	# Stars	Dipole	SASA	DonorHB	AccptHB	QPlogPo/w	QPlogS	QPlogKhsa	# Metab	QPlogBB	%HumOralAbsorption	QPlogHERG	CNS	# RtvFG
Recommended range	(130–725)	(0.0–5.0)	(1–12.50)	(300–1000)	(0–6)	(2.0–20.0)	(−2–6.5)	(−6.5–0.5)	(−1.5–1.5)	(1–8)	(−3–1.2)	(<25% poor; >80% high)	concern below −5	(−2 inactive) (+2 active)	(0–2)
1	170.23	1	2.8	375.85	1	2	2.15	−2.47	−0.34	4	−0.19	83.82	−1.25	−1	-
2	218.33	-	7.35	456.52	-	4	2.1	−1.26	−0.4	6	0.01	75.45	−3.63	1	-
2	218.33	-	7.35	456.52	-	4	2.1	−1.26	−0.4	6	0.01	75.45	−3.63	1	-
3	186.33	5	3.11	416.69	-	0.5	3.73	−4.49	0.38	5	0.61	100	−3.22	2	-
4	310.53	7	0.48	576.92	-	0.5	5.35	−8.24	1.34	10	0.96	100	−3.9	2	-
5	202.33	-	5.95	441.45	-	4	2.01	−1.08	−0.44	6	0.24	78.76	−3.53	1	-
5	202.33	-	5.95	441.45	-	4	2.01	−1.08	−0.44	6	0.24	78.76	−3.53	1	-
6	188.35	5	2.31	409.89	-	1	3.11	−3.82	0.3	3	0.45	100	−2.88	1	-
7	166.19	-	3.67	386.76	1	2	1.96	−1.92	−0.42	2	−0.4	80.8	−2.35	−1	-
8	180.22	3	6.7	417.32	-	1.75	2.21	−2.75	−0.11	3	−0.39	95.04	−4.18	-	-
8	180.22	3	6.61	417.82	-	1.75	2.22	−2.76	−0.11	3	−0.39	95.17	−4.18	-	-
9	230.28	-	3.18	547.88	2	3.4	2.52	−4.13	−0.08	4	−0.8	95.43	−5.93	−1	-
9	230.28	-	3.09	547.44	2	3.4	2.52	−4.12	−0.08	4	−0.8	95.42	−5.92	−1	-
10	214.28	1	2.07	536.85	1	1.7	3.7	−4.76	0.3	4	−0.22	100	−5.9	-	-
11	256.32	-	3.01	596.59	-	2.7	3.92	−5.33	0.4	4	−0.73	100	−6.01	−1	1
11	256.32	-	3.22	600.04	-	2.7	3.91	−5.41	0.4	4	−0.77	100	−6.07	−1	1
12	272.32	-	4.71	639.64	1	3.7	3.61	−5.81	0.34	3	−1.03	100	−6.48	−2	1
12	272.32	-	4.76	640.28	1	3.7	3.61	−5.82	0.34	3	−1.03	100	−6.49	−2	1
13	286.35	-	4.06	664.62	-	3.7	4.35	−6.09	0.44	4	−0.45	100	−6.48	-	1
13	286.35	-	4.29	664.92	-	3.7	4.36	−6.11	0.44	4	−0.44	100	−6.46	-	1
14	290.76	1	5.18	641.98	-	2	5.2	−7.01	0.83	3	−0.19	100	−6.36	-	2
14	290.76	1	5.25	642.37	-	2	5.2	−7.01	0.83	3	−0.18	100	−6.36	-	2
15	248.73	1	5.08	543.19	1	1.7	4.16	−5.53	0.46	3	0.24	100	−5.84	1	-
16	290.76	1	3.84	623.15	-	2	5.14	−6.93	0.85	3	0.1	100	−6.22	1	1
17	398.52	2	2.11	825.87	-	4	6.39	−8.4	1.32	5	−0.97	100	−6.6	−1	2
18	230.28	-	3.09	548.17	2	3.4	2.52	−4.13	−0.08	4	−0.8	95.44	−5.93	−1	-
18	230.28	-	3.07	548.16	2	3.4	2.52	−4.13	−0.08	4	−0.8	95.44	−5.93	−1	-
19	232.3	-	3.57	540.4	2	3.4	2.59	−3.96	−0.05	4	−0.68	100	−5.63	-	-
19	232.3	-	3.49	538.9	2	3.4	2.64	−3.94	−0.06	4	−0.61	100	−5.6	-	-
20	232.3	-	3.7	536.62	2	3.4	2.57	−3.9	−0.06	4	−0.67	100	−5.55	-	-
20	232.3	-	1.64	534.91	2	3.4	2.62	−3.87	−0.06	4	−0.6	100	−5.5	-	-
21	244.31	-	2.9	571.71	1	3.4	3.53	−4.67	0.15	5	−0.27	100	−5.9	-	-
21	244.31	-	2.85	572.08	1	3.4	3.53	−4.68	0.15	5	−0.27	100	−5.91	-	-
22	256.32	-	3.42	600.19	-	2.7	3.91	−5.41	0.4	4	−0.77	100	−6.08	−1	1
22	256.32	-	3.45	600.15	-	2.7	3.91	−5.41	0.4	4	−0.77	100	−6.08	−1	1
23	230.28	-	3.18	547.88	2	3.4	2.52	−4.13	−0.08	4	−0.8	95.43	−5.93	−1	-
24	272.32	-	4.71	639.63	1	3.7	3.61	−5.81	0.34	3	−1.03	100	−6.48	−2	1
25	258.33	-	2.66	619.03	1	3.4	4	−5.37	0.3	5	−0.35	100	−6.24	-	-
26	300.37	2	4.36	710.36	-	3.7	4.83	−6.8	0.61	4	−0.55	100	−6.73	-	1
27	234.31	-	3.3	548.83	-	2.7	3.45	−4.13	0.11	5	−0.82	100	−5.6	−1	1
28	234.31	1	6.27	543.84	-	2.7	3.44	−4.03	0.09	5	−0.77	100	−5.53	−1	1
29	414.47	2	4.68	754.8	6	13.6	-0.11	−2.92	−1.06	8	−3.29	40.05	−5.89	−2	1
29	414.47	2	4.64	734.02	6	13.6	-0.19	−2.69	−1.1	8	−3.13	40.7	−5.7	−2	1
29	414.47	2	5.17	755.35	6	13.6	-0.19	−2.92	−1.08	8	−3.35	39.14	−5.94	−2	1
29	414.47	2	5.73	728.91	6	13.6	-0.23	−2.61	−1.08	8	−3.16	39.41	−5.63	−2	1
30	386.42	-	2.77	688.65	6	13.6	-0.66	−2.47	−1.18	7	−2.88	39.15	−5.64	−2	1
30	386.42	-	3.22	682.74	6	13.6	-0.75	−2.36	−1.15	7	−2.99	36.13	−5.48	−2	1
30	386.42	1	4.11	714.6	6	13.6	-0.74	−2.75	−1.18	7	−3.27	34.82	−5.96	−2	1
30	386.42	1	1.2	710.72	6	13.6	-0.73	−2.7	−1.18	7	−3.22	35.33	−5.91	−2	1
31	232.3	-	2.7	541.33	2	3.4	2.43	−3.82	−0.08	4	−0.79	95.57	−5.54	−1	-
31	232.3	-	2.84	542.23	2	3.4	2.43	−3.84	−0.08	4	−0.8	95.44	−5.56	−1	-
32	264.36	-	1.82	533.75	1	3.4	3.52	−4.3	0.12	5	−0.08	100	−5.49	-	-
32	264.36	-	1.93	538.34	1	3.4	3.54	−4.4	0.13	5	−0.08	100	−5.56	-	-
33	250.33	-	2.38	509.39	2	3.4	2.66	−3.76	−0.09	4	−0.56	96.23	−5.48	-	-
33	250.33	-	2.37	509.81	2	3.4	2.66	−3.77	−0.09	4	−0.56	96.24	−5.49	-	-
34	208.29	-	4.09	422.53	-	2	3.01	−3.34	0.01	2	0.23	100	−4.42	1	-
35	194.27	-	4.61	394.08	-	2	2.75	−2.53	−0.14	2	0.03	100	−4.24	1	-
36	224.29	-	3.29	451.72	-	2	3.15	−3.89	0.14	2	0.14	100	−4.8	1	-
37	216.32	5	0.44	474.39	-	-	5.8	−5.65	0.72	2	0.35	100	−5.56	1	-
38	318.45	1	3.17	674.19	-	2	5.85	−7.32	1.08	4	−0.2	100	−6.41	-	1
39	432.63	9	0.65	807.6	-	-	11.73	−12.03	2.41	6	0.5	100	−8.14	2	-
39	432.63	8	0.93	700.3	-	-	9.78	−11.17	2.17	6	0.52	100	−6.51	2	-
40	432.63	9	0.65	807.6	-	-	11.73	−12.03	2.41	6	0.5	100	−8.14	2	-
40	432.63	8	0.93	700.3	-	-	9.78	−11.17	2.17	6	0.52	100	−6.51	2	-
41	334.45	-	4.91	630.13	1	3.7	4.38	−5.51	0.58	4	−0.77	100	−5.67	−1	1
41	334.45	-	4.93	626.38	1	3.7	4.38	−5.47	0.58	4	−0.74	100	−5.58	−1	1
42	334.45	-	4.82	652.88	1	3.7	4.57	−5.96	0.64	5	−0.76	100	−5.95	−1	1
42	334.45	-	4.4	673.42	1	3.7	4.8	−6.38	0.68	5	−0.64	100	−6.24	-	1
43	234.33	1	2.01	498.72	1	1.7	3.71	−4.39	0.27	4	−0.03	100	−5.45	-	-
44	276.37	-	2.76	592.07	-	2	4.28	−6.08	0.67	3	−0.21	100	−6.15	-	1
45	210.27	-	3.5	401.73	1	2	2.72	−3.04	−0.23	2	−0.22	85.2	−2.48	−1	-
46	388.97	2	4.59	617.73	-	2	6.5	−6.17	1.21	8	0.19	100	−4.68	1	2
46	388.97	1	6.57	609.65	-	2	6.37	−6.02	1.17	8	0.12	100	−4.58	1	2
47	252.35	-	4.52	495.92	1	3.7	2.67	−3.53	−0.07	4	−0.5	96.52	−4.88	-	-
48	278.38	-	2.94	560.83	1	3.4	3.75	−4.74	0.27	6	−0.12	100	−5.36	-	-
49	264.36	-	2.49	532.94	2	3.4	2.98	−4.12	0.04	5	−0.44	100	−5.28	-	-
50	246.34	-	5.04	515.59	-	2	3.88	−4.92	0.41	3	0.1	100	−5.25	1	-
51	248.31	-	4.21	494.76	1	2	3.56	−4.39	0.13	3	−0.42	90.16	−3.34	−1	-
52	232.32	-	5.54	487.09	-	2	3.35	−4.29	0.26	3	−0.13	100	−5.11	-	-
53	234.33	1	1.8	496.6	1	1.7	3.64	−4.49	0.3	4	0.05	100	−5.21	1	-
54	248.31	-	4.21	495.1	1	2	3.57	−4.39	0.13	3	−0.42	90.17	−3.35	−1	-
55	262.34	-	4.22	544.7	-	2	4.12	−5.47	0.54	3	0	100	−5.55	-	-
56	304.42	4	1.89	591.79	-	1.5	5.59	−6.89	1.06	4	0.15	100	−5.48	1	1
56	304.42	4	1.89	591.47	-	1.5	5.58	−6.9	1.07	4	0.15	100	−5.46	1	1
57	278.34	1	2.8	547.2	2	5.4	1.7	−3.68	−0.27	4	−1.47	77.15	−5.44	−2	-
57	278.34	1	3.43	547.55	2	5.4	1.7	−3.69	−0.27	4	−1.47	77.15	−5.45	−2	-
58	266.37	-	4.99	531.75	1	3.7	2.98	−4.13	0.1	5	−0.56	100	−4.91	-	-
59	294.34	1	1.92	555.27	3	5.4	1.93	−3.98	−0.45	4	−1.78	62.81	−3.68	−2	-
59	294.34	1	2.76	555.53	3	5.4	1.93	−3.98	−0.45	4	−1.78	62.81	−3.68	−2	-
60	248.36	1	2.19	534.06	1	1.7	4.03	−5.03	0.44	5	−0.06	100	−5.44	-	-
61	298.37	-	5.85	542.89	3	7.1	1.07	−2.98	−0.5	5	−1.4	74.44	−4.8	−2	-
61	298.37	-	4.88	542.6	3	7.1	1.06	−2.98	−0.5	5	−1.4	74.36	−4.79	−2	-
62	366.92	3	3.08	712.65	-	2	6.67	−8.41	1.37	5	0.21	100	−6.17	1	1
63	432.59	4	5.23	855.53	-	4	7	−9.13	1.52	6	−0.86	100	−6.63	−1	2
64	282.8	1	5.1	546.49	1	1.7	4.64	−5.5	0.57	4	0.34	100	−5.39	1	-
65	348.47	-	4.77	685.58	1	3.7	4.9	−6.42	0.76	5	−0.85	100	−5.81	−1	1
66	348.47	1	5.79	721.29	1	3.7	5.08	−7.07	0.82	6	−0.93	100	−6.37	−1	1
67	678.93	10	5.34	1180.96	-	5.7	10.73	−13.34	2.73	11	−1.63	100	−8.35	−2	2
68	492.68	5	2.25	916.97	-	3.4	9.48	−11.13	2.07	8	−0.68	100	−8.28	-	1
69	492.68	5	2.32	856.86	-	3.4	8.46	−10.47	1.88	8	−0.68	100	−7.68	-	1
70	492.68	6	1.67	848.1	-	3.4	8.4	−10.35	1.93	10	−0.68	100	−7.54	-	-
71	364.47	-	4.17	731.03	2	5.4	3.97	−6.33	0.43	6	−1.58	95.78	−6.38	−2	1
72	262.34	1	4.69	537.04	1	3.7	2.72	−4.24	0.05	4	−0.92	91.26	−5.42	−1	-
73	246.34	1	2.19	521.45	1	1.7	3.88	−4.78	0.38	3	−0.03	100	−5.63	-	-
74	264.36	-	2.47	545.82	2	3.4	2.92	−4.18	0.02	5	−0.73	96.95	−5.5	−1	-
75	248.38	6	0.96	464.31	-	-	5.71	−7.4	0.83	3	0.19	100	−5.33	1	-
76	276.39	1	4.61	502.07	-	2	3.95	−4.98	0.46	3	0.03	100	−5.34	1	-
77	278.4	1	2.58	511.2	1	1.7	4.22	−5.06	0.5	4	0.2	100	−5.44	1	-
78	278.4	3	1.84	505.75	-	0.75	5.62	−5.96	0.83	4	0.03	100	−5.34	1	-
79	360.5	1	2.73	671.68	-	2	6.27	−7.84	1.29	6	0.18	100	−6.45	1	1
80	360.5	1	3.82	678.33	-	2	6.24	−7.98	1.3	6	0.08	100	−6.53	1	1
81	292.43	2	2.1	542.23	-	1.7	5.44	−5.81	0.72	5	−0.31	100	−5.61	-	-
82	426.52	-	3.15	644.26	4	9.25	2.04	−4.08	−0.27	7	−1.16	83.97	−5.71	−2	1
82	426.52	-	3.4	642.05	4	9.25	2.03	−4.08	−0.26	7	−1.21	82.47	−5.59	−2	1
82	426.52	-	4.89	649.11	4	9.25	2.05	−4.16	−0.26	7	−1.21	83.16	−5.76	−2	1
82	426.52	-	2.93	642.67	4	9.25	2.16	−4.08	−0.27	7	−0.99	87.19	−5.61	−1	1
83	540.79	6	4.22	945.88	-	2	9.78	−12.95	2.68	6	−0.12	100	−8.05	-	-
84	197.25	-	6.72	419.53	1	4.7	1.09	−2.13	−0.51	4	−0.42	86.7	−3.64	-	-
85	316.37	1	3.62	593.36	2	5.45	2.42	−4.27	0.05	6	−1.29	85.52	−5.02	−2	1
86	280.38	-	4.82	549.83	1	5.4	2.64	−3.42	−0.09	5	−0.73	95.86	−4.44	−1	-
86	280.38	-	6.54	568.11	1	4.45	3.19	−4.05	0.12	4	−0.68	100	−4.85	-	-
86	280.38	-	6.55	555.35	1	4.45	3.06	−3.82	0.08	4	−0.71	100	−4.68	−1	-
86	280.38	-	5.66	564.44	1	4.45	3.08	−4.01	0.09	4	−0.75	100	−4.83	−1	-
86	280.38	-	4.24	564.71	1	4.45	3.08	−3.98	0.11	4	−0.79	100	−4.78	−1	-
87	350.41	-	9.97	600.63	1.25	6.25	3.04	−4.55	−0.14	3	−1.21	76.31	−3.41	−2	1
87	350.41	-	3.36	593.33	1.25	6.25	2.95	−4.37	−0.14	3	−1.23	75.36	−3.25	−2	1
87	350.41	-	7.76	582.94	1.25	6.25	2.99	−4.17	−0.17	3	−1.04	78.73	−3.14	−2	1
87	350.41	-	8.61	596.55	1.25	6.25	3.04	−4.47	−0.14	3	−1.18	76.64	−3.33	−2	1
88	432.58	2	2.52	761.06	1	3.75	6.09	−7.38	1.59	3	0.48	100	−6.96	2	-
88	432.58	2	2.52	761.06	1	3.75	6.09	−7.38	1.59	3	0.48	100	−6.96	2	-
89	332.41	-	3.73	648.07	-	4.75	4.13	−5.57	0.45	3	−0.34	100	−5.16	-	-
90	376.42	-	5.82	693.39	1	6.75	3.51	−5.75	0.2	3	−1.6	76.93	−3.34	−2	-
91	348.41	-	2.67	673.35	1	6.45	3.34	−5.55	0.28	3	−0.88	100	−5.43	−1	-
92	348.41	-	5.79	664.93	1	6.45	3.2	−5.39	0.28	3	−1.03	94.16	−5.22	−2	-
93	350.43	-	2.82	679.38	1	6.45	3.41	−5.5	0.28	4	−0.95	100	−5.29	−1	-
94	350.43	-	4.86	665.38	1	6.45	3.35	−5.25	0.26	4	−0.91	100	−5.09	−1	-
95	234.31	-	6.4	449.04	-	3.7	2.41	−2.95	−0.18	5	0.28	100	−3.85	1	-
95	234.31	-	6.3	424.55	-	2.75	2.8	−3	0.03	3	0.3	100	−3.42	1	-
95	234.31	-	6.52	444.21	-	2.75	2.96	−3.37	0.1	3	0.26	100	−3.77	1	-
95	234.31	-	6.52	444.21	-	2.75	2.96	−3.37	0.1	3	0.26	100	−3.77	1	-
95	234.31	-	6.3	424.54	-	2.75	2.8	−3	0.03	3	0.3	100	−3.42	1	-
96	294.37	-	6.75	555.68	1	5.7	2.37	−3.57	−0.1	5	−0.9	90.35	−4.57	−1	1
96	294.37	-	4.21	564.47	1	6.2	2.14	−3.54	−0.17	4	−1.09	85.98	−4.81	−2	1
96	294.37	-	8.99	556.37	1	6.2	2.17	−3.43	−0.18	4	−0.96	87.87	−4.63	−1	1
96	294.37	-	4.8	551.9	1	6.2	2.08	−3.35	−0.2	4	−1.03	86.02	−4.63	−2	1
96	294.37	-	7.82	549	1	6.2	2.16	−3.29	−0.22	4	−0.88	89.33	−4.65	−1	1

# Stars: Number of property or descriptor values that fall outside the 95% range of similar values for known drugs. Many stars suggest that a molecule is less drug-like than molecules with few stars; Dipole: Computed dipole moment of the molecule; SASA: Total solvent accessible surface area (SASA) in square angstroms using a probe with a 1.4 Å radius; Donor H-bond: Estimated number of hydrogen bonds that the solute would donate to water molecules in an aqueous solution; Acceptor H-bond: Estimated number of hydrogen bonds that the solute would accept from water molecules in an aqueous solution; QPlogPo/w: Predicted octanol/water partition coefficient.; QPlogS: Predicted aqueous solubility, log S; QPlogkhsa: Prediction of binding to human serum albumin; # Metab: Number of likely metabolic reactions.; QplogBB: Predicted brain/blood partition coefficient; % Human Oral Absorption: Predicted human oral absorption on 0 to 100% scale; QPlogHERG: Predicted IC50 value for blockage of HERG K+ channels; CNS: Predicted central nervous system activity on a −2 (inactive) to +2 (active) scale; # rtvFG: Number of reactive functional groups; the specific groups are listed in the jobname out file. The presence of these groups can lead to false positives in HTS assays and to decomposition, reactivity, or toxicity problems in vivo.

5.2. Ligands and Proteins Preparations

Using LigPrep converted 2D structures to 3D, tautomerization, and ionization gave 146 minimized 3D structures that were utilized for docking with the Cathepsin D crystal structure (PDB: 4OD9). The 4OD9 prepared by the protein preparation wizard tool minimized the geometry and optimized the H-bond network. Specifying the proper force field treatment and the formal charge was accomplished by the addition of correct ionization states and missing hydrogens (Figure 12).

Figure 12

N-(3,4-dimethoxybenzyl)-Nalpha-{N-[(3,4-dimethoxyphenyl)acetyl] carbamimidoyl}-D-phenylalaninamide (2RZ) complexed with Cathepsin D PDB: 4OD9 after preparation, using Protein Preparation Wizard, from Schrodinger, where hydrogen bonds are optimized, and the whole complex minimized and represented as a 3D structure.

5.3. Molecular Docking Studies

After designating the grid box in the prepared protein through Glide’s Receptor-Grid Generation tool in Maestro [78], the obtained 3D molecular structures were docked into the cathepsin D co-crystallized inhibitor binding site. Table 5 shows the results of the docked ligands that were selected owing to their most negative docking scores. These scores demonstrated the best-bonded ligand relative binding affinities and conformations. Compounds 29 and 30 displayed the highest negative docking scores of −9.439 and −9.178 kcal/mol in complex with 4OD9, respectively, while the reference inhibitors (2RZ) had a score of −6.895 kcal/mol in complex with the same protein.

Table 5

In silico screening results of thiophene derivatives against Cathepsin D (PDB: 4OD9).

Compd	Docking Score	XP GScore	Glide GScore	Glide Emodel
29	−9.439	−9.439	−9.439	−62.9
30	−9.178	−9.178	−9.178	−61.357
82	−8.09	−8.09	−8.09	−64.014
42	−6.971	−6.971	−6.971	−45.831
Ref_4OD9	−6.895	−7.306	−7.306	−71.557
66	−6.889	−6.889	−6.889	−56.795
64	−6.772	−6.772	−6.772	−47.815
61	−6.677	−6.677	−6.677	−45.925
39	−6.567	−6.567	−6.567	−68.455
40	−6.567	−6.567	−6.567	−68.455
57	−6.484	−6.484	−6.484	−44.895
33	−6.478	−6.478	−6.478	−37.519
46	−6.385	−6.385	−6.385	−54.121
19	−6.374	−6.374	−6.374	−39.754
81	−6.373	−6.373	−6.373	−40.88
92	−6.297	−6.297	−6.297	−50.1
9	−6.119	−6.119	−6.119	−37.184
52	−6.056	−6.056	−6.056	−39.105
2	−5.946	−5.946	−5.946	−35.558
89	−5.913	−5.913	−5.913	−41.976
68	−5.91	−5.91	−5.91	−58.146
59	−5.879	−5.879	−5.879	−43.627
58	−5.789	−5.789	−5.789	−39.479
41	−5.775	−5.778	−5.778	−50.687
84	−5.761	−5.761	−5.761	−35.037
31	−5.624	−5.624	−5.624	−39.484
17	−5.589	−5.589	−5.589	−58.566
91	−5.563	−5.563	−5.563	−39.688
65	−5.534	−5.534	−5.534	−52.028
74	−5.522	−5.522	−5.522	−50.032
49	−5.439	−5.439	−5.439	−37.501
53	−5.431	−5.431	−5.431	−42.062
60	−5.413	−5.413	−5.413	−48.483
32	−5.359	−5.359	−5.359	−36.918
37	−5.344	−5.344	−5.344	−38.255
95	−5.323	−5.733	−5.733	−31.554
12	−5.317	−5.32	−5.32	−48.638
93	−5.294	−5.294	−5.294	−41.169
71	−5.234	−5.234	−5.234	−56.25
77	−5.182	−5.182	−5.182	−47.296
80	−5.175	−5.175	−5.175	−52.89
20	−5.032	−5.032	−5.032	−38.495
47	−4.988	−4.988	−4.988	−41.313
48	−4.959	−4.959	−4.959	−45.598
79	−4.959	−4.959	−4.959	−53.558
87	−4.892	−4.892	−4.892	−42.972
70	−4.788	−4.788	−4.788	−66.838
16	−4.761	−4.761	−4.761	−39.898
22	−4.708	−4.708	−4.708	−43.587
76	−4.681	−4.681	−4.681	−42.185
50	−4.616	−4.616	−4.616	−42.266
62	−4.615	−4.615	−4.615	−53.205
38	−4.587	−4.587	−4.587	−44.277
21	−4.479	−4.479	−4.479	−34.302
96	−4.434	−4.844	−4.844	−43.679
27	−4.427	−4.427	−4.427	−32.461
54	−4.4	−4.4	−4.4	−42.328
36	−4.372	−4.372	−4.372	−31.206
34	−4.367	−4.367	−4.367	−32.958
10	−4.36	−4.36	−4.36	−37.183
4	−4.325	−4.325	−4.325	−44.054
55	−4.289	−4.289	−4.289	−41.301
86	−4.263	−4.673	−4.673	−34.699
67	−4.242	−4.242	−4.242	−71.461
72	−4.221	−4.221	−4.221	−45.333
56	−4.166	−4.166	−4.166	−45.866
18	−4.159	−4.159	−4.159	−38.902
28	−4.131	−4.131	−4.131	−38.886
51	−4.127	−4.127	−4.127	−38.518
73	−4.109	−4.109	−4.109	−41.377
88	−4.09	−4.09	−4.09	−46.666
94	−4.085	−4.085	−4.085	−40.186
35	−4.079	−4.079	−4.079	−30.767
44	−4.047	−4.047	−4.047	−41.625
75	−4.012	−4.012	−4.012	−39.751
69	−3.964	−3.964	−3.964	−65.916
85	−3.899	−3.899	−3.899	−35.518
83	−3.778	−3.778	−3.778	−63.675
13	−3.769	−3.769	−3.769	−38.792
9	−3.688	−3.688	−3.688	−41.071
23	−3.688	−3.688	−3.688	−41.071
14	−3.65	−3.65	−3.65	−38.638
11	−3.64	−3.64	−3.64	−42.16
43	−3.528	−3.528	−3.528	−37.644
25	−3.481	−3.481	−3.481	−39.665
24	−3.48	−3.483	−3.483	−44.761
78	−3.406	−3.406	−3.406	−37.385
45	−3.403	−3.403	−3.403	−30.006
3	−3.303	−3.303	−3.303	−26.546
15	−3.253	−3.253	−3.253	−35.921
90	−3.239	−3.239	−3.239	−43.733
26	−3.185	−3.185	−3.185	−40.847
8	−2.058	−2.117	−2.117	−23.999
63	−1.85	−1.85	−1.85	−70.342
7	−1.55	−1.55	−1.55	−18.635
5	−0.841	−0.841	−0.841	−10.861

Analysis of the docking of 29 and 30 compared with the redocked reference 2RZ indicated that they interacted through hydrogen bonds (Figure 13, Figure 14, Figure 15 and Figure 16) with the binding site residues of Cathepsin D (4OD9). The binding site residues VAL 31, ASN 38, and TRP 40 of Cathepsin D had hydrogen bonding with the different hydroxyl groups of the sugar part. TRY 16 interacted with the terminal hydroxy group of 29. Whilst the binding site residues VAL 31, ASP 33, and TRP 40 of Cathepsin D possessed hydrogen bonding with the various OH groups of sugar part, and ASP 50 interacted with the terminal hydroxy group of the 30.

Figure 13

Cathepsin D in complex with 29 represented as 3D molecular surface and ribbon structure.

Figure 14

(A) Putative binding mode of 29 in the binding site of cathepsin PDB: 4OD9. Compound 29 is displayed as green sticks. The amino acids residues of the binding site are represented as grey sticks, and H-bonds are represented in yellow dotted, (B) 2D depiction of the ligand–protein interactions.

Figure 15

(A) Putative binding mode of compound 30 in the binding site of Cathepsin PDB: 4OD9. Compound 30 is displayed as green sticks. The amino acids residues of the binding site are represented as grey sticks, and H-bonds are represented in yellow dotted, (B) 2D depiction of the ligand–protein interactions.

Figure 16

(A) Putative binding mode of Reference in the binding site of Cathepsin PDB: 4OD9. The green sticks represent the Reference, whereas the grey sticks represent amino acid residues, and the yellow dotted lines represent the H-bond. (B) 2D depiction of the ligand–protein interactions.

5.4. Molecular Dynamics Simulation

The docking operation is a static view for the molecule’s binding in the active site of the specific protein. MD simulation computes the time versus atoms motions. By using Desmond software [79,80,81], the stability and frequency of compound 30 complex with Cathepsin D with PDB codes 4OD9, MD simulation was run with simulation time 100 ns. The complex structure was optimized at pH 7.0 ± 2.0. Complex stability was examined by analyzing the interaction map and the RMSD (root mean square deviation) plots of the ligand and protein. The RMSD plot in Figure 17 for the compound 30 complexed with Cathepsin D indicated the complexes tend to stabilize during simulation (100 ns) with regard to a reference frame at time 0 ns. There was a slight fluctuation during the simulation, but it lay under the permitted range of 1–3 Å; hence, it can be regarded as non-significant. Since the RMSD plots of compound 29 and protein backbone were lying over each other, the stable complex formation can be inferred. Figure 18 showed the schematic of detailed ligand atom interactions of compound 30 with Cathepsin D. The docked poses were maintained through the simulation time of 100 ns, i.e., molecular interactions with residues VAL 31, SER 36, ASN 38, TRP 40, and TYR 78.

Figure 17

RMSD analysis for compound 30 complexed with cathepsin D (PDB Code) of MD simulation trajectory. The RMSD plot was obtained for compound 30 complexed with cathepsin D (PDB ID 4OD9). The 100 ns simulation time reaffirms the stability of the complex without any significant changes in the structure.

Figure 18

Cathepsin D interactions with compound 30 throughout the simulation. The interactions between the ligand and protein are classified into hydrophobic, ionic, hydrogen bond, and water bridges. Each classification can be further sub-grouped and observed in the ‘Simulation Interactions Diagram’ panel. The stacked bar charts are normalized throughout the trajectory. The stacked bar charts are normalized over the course of the trajectory: for example, a value of 0.7 suggests that 70% of the simulation time, the specific interaction is maintained. Values over 1.0 are possible, as some protein residue may make multiple contacts of the same subtype with the ligand.

Figure 19 represents the ligand–protein interactions that are characterized into four types: ionic, hydrophobic, hydrogen bonds, and water bridges. Each interaction type includes more specified subtypes, which can be investigated via the ‘Simulation Interactions Diagram’ panel [82,83,84,85,86]. The stacked bar charts were normalized throughout the trajectory: for example, a value of 0.7 suggests that 70% of the simulation time, the specific interaction, is maintained. Values over 1.0 are possible, as some protein residue may make multiple contacts of the same subtype with the ligand. Hydrogen bonding with residues VAL 31, TRP 40, and TYR 78 was retained for more than 80% of the simulation time.

Figure 19

The schematic diagram shows the detailed atomic interaction of compound 30 with Cathepsin D. Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns). It is possible to have interactions with >100% as some residues may have multiple interactions of a single type with the same ligand atom.

5.5. Materials and Methods

5.5.1. Preparation of PDB Structures

The PDB structure (PDB IDs: 4OD9) was downloaded from the Protein Data Bank [69], prepared and optimized utilizing the “Protein preparation wizard” [70] tool of Schrödinger suite [76,87]. For this reason, the bond orders for known HET groups and untemplated residues were identified, and hydrogens were added. Then, breaking bonds to metals, adding zero-order bonds among metals and nearby atoms, and correcting the formal charges to metals and neighboring atoms were carried out. From HET groups, water molecules beyond 5 Å were deleted. Disulfide bonds were generated. For ligands, metal HET states and cofactors were generated at 7.0 ± 2.0 pH using LigPrep [76]. Finally, H-bonds optimization at pH 7.0 using PROPKA [88], the removal of water molecules beyond 3 Å from HET groups, and restrained minimization using the OPLS4 force field were done.

5.5.2. ADME Properties Prediction

The drug likeness and ADME properties of the chosen compounds were estimated via the Maestro Schrodinger QikProp module in terms of metabolism, distribution, excretion, absorption, etc. [77].

5.5.3. Receptor Grids Generation and Docking

Glide [78] was utilized for both grid generation and ligands docking. For docking of the 96 thiophene derivatives, the grid was generated using the PDB: 4OD9, the region of binding was specified by selecting 2RZ. The non-polar atoms were set for the VdW radii scaling factor by 1.0 and the partial charge cut-off 0.25. The ligands docking was performed using the “ligand docking” tool of the Schrödinger suite [78,85]. The selected protocol was standard precision (SP), the ligand sampling method was flexible, and all the other settings were default.

5.5.4. MD Simulations of Compound 30 in Complex with 4OD9

MD simulations were run using the Schrödinger suite [88]. The system of compound 30 in complex with 4OD9 was retrieved from the docking results and first tuned through the “System Builder” tool. The TIP3P solvent model and then orthorhombic-shape box shape was chosen. The system was neutralized by adding Na+ ions, and the side distances box was set at 10 Å. The MD calculations were run for 100 ns per trajectory, the number of atoms, pressure, and temperature were kept constant (NPT ensemble). In contrast, the pressure was set at 1.01325 bar and temperature at 300.0 K, and the force field was set as OPLS4.

6. Conclusions

Natural products are featured by enormous scaffold diversity and structural complexity that contribute to drug discovery. Sulfur-containing natural metabolites are a large class of significant functional molecules with potent biological activities and pharmacological properties; some of them have been developed into essential drugs. In the current work, 96 naturally occurring thiophenes have been reported from 2015 till now. Most of them had one to three thiophene rings. However, dimeric bithiophenes with four thiophene rings and quinquethiophenes with five thiophene rings were extremely rare. These metabolites have been mainly evaluated for their cytotoxic, antimicrobial, anti-inflammatory, and nematicidal capacities. On the other side, there are limited reports on their antimalarial, larvicidal, antioxidant, and anti-influenza activities. Some of them showed remarkable cytotoxic effects. Therefore, they could be a potential therapeutic agent for treating various cancers. However, in vivo studies and their detailed mechanism of action need to be determined.

Furthermore, they showed marked antifungal potential against various plant pathogenic fungi and had a remarkable nematicidal effect. Hence, they could provide worthy insights for discovering effective, eco-friendly nematicides and fungicides. More studies on formulations development are needed to upgrade stability and efficacy and cut down costs. Additionally, field assessment and research on the effects of these compounds on non-target organisms are compulsory. Their antifungal mechanism was proposed to be photodynamic. However, further assessments are needed to confirm their potential for application as photosensitizers in photodynamic antimicrobial chemotherapy toward fungal infections. The structure–activity relationship study revealed that the presence of the acetylene group in the side chain increased the larvicidal and antimalarial potential. However, the attachment of the acetylenic side chain to an ester and acyl functional groups lowered the activity [19,32,34,41]. Additionally, increasing the number of acetylene groups in the side chain increased the anti-inflammatory, nematocidal, and antifungal capacities [19,32,34,41]. Further, the attachment of this chain with the chlorine group enhanced the activity [19] (Figure 20). Estimation of other potential bioactivities and derivatization of these metabolites, besides the mechanistic and in vivo studies of these metabolites, should be the target of future research. The precursor-based combinatorial biosynthesis (PCB) could be used for enhancing the production and structural modification of these metabolites. In this technique, a precursor analog has to be fed to the producing microorganism, resulting in the production of novel thiophenes with a potential pharmaceutical significance that is effective and ecologically friendly [89].

Figure 20

Summary of the reported structure–activity relationship (SAR) of thiophenes. ↑: increase; ↓: decrease [19,32,34,41].

Based on the in silico, including ADMET properties predication, molecular docking for the protein–ligands binding interaction, and molecular dynamics, these metabolites were identified as potential inhibitors for the Cathepsin D, which will be helpful as potential leads for the treatment of several diseases that are affected by the dysregulation of this enzyme. The results of the studies described in this review are undoubtedly significant. They constitute the first stage of searching for new drug candidates and observing the strength of the tested metabolites concerning the currently used drugs. Furthermore, this review provides an overview of the research progress on naturally isolated thiophenes with special highlighting of their bioactivities that could attract the attention of many natural product researchers to further investigate and explore their mechanism, efficacy, and safety through in vivo studies. The structural diversity of thiophenes could be of considerable synthetic interest as novel chemical entities for drug discovery. Also, this work may encourage further research for isolation, characterization, and bio-evaluation of these thiophenes that may provide more candidates for the pharmaceutical industry. Additionally, it aims at providing a reference for researchers that they can use for the rapid identification of isolated thiophenes through a comparison of their physical and spectral data. Future investigations such as combinatorial chemistry and drug design will inevitably expose new avenues for the advancement of drug discovery.