Naturally occurring thiophenes: isolation, purification,structural elucidation, and evaluation of bioactivities

Sabrin R. M. Ibrahim • Hossam M. Abdallah •

Ali M. El-Halawany • Gamal A. Mohamed

Received: 26 December 2014 / Accepted: 17 March 2015 / Published online: 22 March 2015

� Springer Science+Business Media Dordrecht 2015

Abstract Thiophenes are a class of heterocyclic

aromatic compounds based on a five-membered ring

made up of one sulfur and four carbon atoms. The

thiophene nucleus is well established as an interesting

moiety, with numerous applications in a variety of

different research areas. Naturally occurring thio-

phenes are characteristic secondary metabolites

derived from plants belonging to the family Aster-

aceae, such as Tagetes, Echinops, Artemisia, Bal-

samorhiza, Blumea, Pluchea, Porophyllum and

Eclipta. Furthermore, naturally occurring thiophenes

are generally composed of one to five thiophene rings

that are coupled together through their a-carbons, andcarry alkyl chains on their free ortho-positions.

Thiophene-containing compounds possess a wide

range of biological properties, such as antimicrobial,

antiviral, HIV-1 protease inhibitor, antileishmanial,

nematicidal, insecticidal, phototoxic and anticancer

activities. This review focuses on naturally occurring

thiophene derivatives; their sources, physical and

spectral data, and biological activities.

Keywords Thiophenes � Biosynthesis � NMR data �Anti microbial � Cytotoxic

Introduction

Thiophenes are a class of heterocyclic aromatic

compounds based on a five membered ring containing

one sulfur and four carbon atoms with a molecular

formula of C4H4S. The word ‘thiophene’ is derived

from the Greek words ‘theion’ and ‘phaino’, which

mean sulfur and shining, respectively. Thiophene

derivatives make up a significant proportion of the

organosulfur-containing compounds found in petro-

leum, as well as several other products derived from

fossil fuels, and are formed as the by-products of

petroleum distillation (Chaudhary et al. 2012; Mishra

et al. 2011). Natural thiophenes are characteristic

secondary metabolites of plants belonging to the

S. R. M. Ibrahim

Department of Pharmacognosy and Pharmaceutical

Chemistry, Faculty of Pharmacy, Taibah University,

Al Madinah Al Munawwarah 30078, Kingdom of Saudi

Arabia

S. R. M. Ibrahim

Department of Pharmacognosy, Faculty of Pharmacy,

Assiut University, Assiut 71526, Egypt

H. M. Abdallah � A. M. El-Halawany � G. A. Mohamed

Department of Natural Products and Alternative

Medicine, Faculty of Pharmacy, King Abdulaziz

University, Jeddah 21589, Kingdom of Saudi Arabia

H. M. Abdallah � A. M. El-Halawany (&)

Department of Pharmacognosy, Faculty of Pharmacy,

Cairo University, Cairo 11562, Egypt

e-mail: ahalawany2003@yahoo.com

G. A. Mohamed

Department of Pharmacognosy, Faculty of Pharmacy,

Al-Azhar University, Assiut Branch, Assiut 71524, Egypt

123

Phytochem Rev (2016) 15:197–220

DOI 10.1007/s11101-015-9403-7

family Asteraceae, including the following genera:

Tagetes, Echinops, Artemisia, Balsamorhiza, Blumea,

Pluchea, Porophyllum, and Eclipta. Thiophene

derivatives isolated from natural sources can be

classified according to the number of thiophene rings

in their structure, including thiophenes (one ring),

bithiophenes (two rings), terthiophenes (three rings)

and quinquethiophenes (five rings) (Fig. 1). Thio-

phene and its derivatives are produced as part of the

chemical defense mechanism in numerous plant

species, which involve the manufacture and storage

of organic substances in different parts of the plants.

These compounds can behave as repellents, act as

toxic substances or have anti-nutritional effects on

herbivores (Gil et al. 2002). Natural thiophenes are

derived from polyacetylenes, which can be stored in

plant tissues or released into the soil (Tang et al. 1987).

These compounds can also act as toxins that are

activated by sunlight or UV irradiation (300–400 nm).

These compounds are toxic towards numerous patho-

gens, including nematodes, insects, fungi, and bacteria

(Champagne et al. 1984; Gil et al. 2002).

A recent review of the available literature revealed

that there are currently no reviews pertaining to the

biosynthesis, isolation and biological activity of

naturally occurring thiophenes. Herein, we have listed

the thiophenes that have been reported in the literature

over the past few decades and provided a summary of

their biological activities, physical constants, spectral

data, plant sources, and associated references. These

data have been listed in the following order for each

compound: name, structure, melting point (�C), opti-cal rotation (concentration, solvent), UV (solvent,

kmax nm, log e), IR (medium, absorption band in

cm-1), 1H NMR (spectrometer frequency, solvent,

chemical shift values in d ppm), 13C NMR (spec-

trometer frequency, solvent, chemical shift in dvalues), plant source (family), molecular formula,

calculated molecular weight and reference(s). The 1H

and 13C NMR data have been rounded to two and one

decimal places, respectively. The molecular weight

data have been rounded to four decimal places. The

NMR data have been listed on each structure because

of the differences in the systems used to number the

different structures. The principle aim of this review is

to provide a reference for researchers that they can use

for the rapid identification of isolated thiophenes

through a comparison of their physical and spectral

data. The highlighted bioactivities of these compounds

may also be of interest to synthetic and medicinal

chemists for the design of new drugs using known

thiophenes as raw materials. The thiophenes described

in this review have been arranged in five different

groups according to the number of thiophene rings in

their structure, including group I-thiophene, group II-

bithiophenes, group III-terthiophenes, group IV-quin-

quethiophenes, and group V-miscellaneous thio-

phenes (Tables 1, 2, 3, 4, 5).

Thiophene biosynthesis

The first naturally occurring thiophene derivative, a-terthiophene, was isolated in 1947 from Tagetes erecta

(Zechmeister and Sease 1947). Since then, more than

150 thiophene-based natural products comprising one,

two or three thiophene rings and side chains bearing a

variable number of double or triple bonds (Bohlmann

and Zdero 1985; Kagan 1991) had been characterized

from Asteraceae and fungi (Bohlmann 1988; Sorensen

Fig. 1 Classes of naturally

occurring thiophenes

198 Phytochem Rev (2016) 15:197–220

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Table 1 Naturally occurring thiophene: group-I: thiophene

1. 3-(4,8,12,16-Tetramethylheptadeca-3,7,11,15-tetraenyl)-thiophene-1-oxide

Pale yellowish oil; UV kmax (CH3OH) (log e): 218 (4.42) nm; IR (Nujol) cmax: 2925, 1642, 1230, 1025 cm-1; EIMS m/z (rel. int.):

386 [M]? (10), 371 (17), 315 (18), 293 (7), 285 (15), 272 (5), 217 (8), 204 (15), 175 (10), 161 (12), 149 (17), 147 (10), 135 (27),

123 (22), 95 (27), 81 (94), 69 (100); HREIMS m/z: 386.2643 (calcd. for C25H38OS, 386.2645); NMR data (CDCl3, 500 and

125 MHz); The marine sponge Xestospongia sp. (Pedpradab and Suwanborirux 2011)

2. Xanthopappin A; 2-(E)-Hept-5-ene-1,3-diynylthiophene diol

Brown oil; UV kmax (CH3OH) (log e): 206 (4.31), 252 (4.22), 313 (4.08) nm; EIMS m/z (rel. int.): 172 [M]? (47), 171 [M–H]? (34),

144 [M–C2H4]? (32); HRTOFMS m/z: 173.0428 [M?H]? (calcd. for C11H9S, 173.0424); NMR data (CDCl3, 500 and 125 MHz);

The stems and roots of Xanthopappus subacaulis C. Winkl (family: Asteraceae) (Tian et al. 2006)

3. 10,11-Threo-xanthopappin D; 2-Hept-5,6-threo-dihydroxy-1,3-diynylthiophene

Colourless oil; [a]D -20 (c 0.5, acetone); UV kmax (CH3OH): 306, 290, 232 nm; IR (KBr) cmax: 3367 (OH), 2924 (CH3), 2233

(C:C) cm-1; HRESIMS m/z: 229.0292 [M?Na]? (calcd. for C11H10O2SNa, 229.0294); NMR data (CDCl3, 600 and 150 MHz);

Whole plant of Xanthopappus subacaulis C. Winkl (family: Asteraceae) (Zhang et al. 2014)

4. 10,11-Erythro-xanthopappin D; 2-Hept-5,6-erythro-dihydroxy-1,3-diynylthiophene

Colourless oil; [a]D ?20 (c 0.5, acetone); UV kmax (CH3OH): 304, 289, 232 nm; IR (KBr) cmax: 3345 (OH), 2924 (CH3), 2219

(C:C) cm-1; HRESIMS m/z: 435.0687 [2M?Na]? (calcd. for 2(C11H10O2S) Na, 435.0695); NMR data (CDCl3, 600 and

150 MHz); Whole plant of Xanthopappus subacaulis C. Winkl (family: Asteraceae) (Zhang et al. 2014)

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Table 1 continued

5. N-Isobutyl-6-(2-thiophenyl)-2,4-hexadienamide

HRESIMS m/z: 272.1072 ([M?Na]?, (calcd. for C14H19NOS); NMR (CDCl3, 300 and 75 MHz); Leaves of Chrysanthemum

coronarium L. (family: Asteraceae) (Ragasa et al. 1997)

6. Amplectol; (3,4-Dihydroxy-8-[50-methyl-thiophen-20-yl]-1,5-octadien-7-yne)

Colorless oil; UV kmax (CH3OH): 295 nm; IR mmax: 3620, 3565 (OH), 2200 (C:C) cm-1; HREIMS m/z (rel. int.): 234.072 [M]?

(calcd. for C13H14O2S, 234.073) (6), 216 [M–H2O]? (9), 177 [(M–CH(OH)CH=CH2)]

?; NMR data (CDCl3, 400 MHz); Aerial

parts of Blumea amplectens DC var. arenaria (family: Asteraceae) (Pathak et al. 1987)

7A. Echinoynethiophene A; 7,10-Epithio-7,9-tridecadiene-3,5,11-triyne-1,2-diol

Yellow needles (acetone), mp. 122–123 �C; IR (KBr) cmax: 3328 (br), 3104, 2956, 2923, 2872, 2150, 1778, 1451, 1322, 1186, 1080

(s), 1022, 946, 864, 805, 688 cm-1; C13H10O2S; EIMS m/z (rel. int.): 230 [M]? (90), 199 (100), 171 (33), 170 (32), 169 (33), 145

(22), 139 (20), 127 (50); NMR data (Acetone-d6, 500 and 125 MHz); Roots of Echinops grijissii Hance (family: Asteraceae) (Liu

et al. 2002)

7B. Echinoynethiophene A; 7,10-Epithio-7,9-tridecadiene-3,5,11-triyne-1,2-diol

Yellow amorphous powder; [a]D ?92.2 (c 0.1 CH3OH); UV kmax (e): 237 (7682), 245 (10,293), 251 (10,293), 273 (7728), 275

(7935), 280 (8556), 324 (17,917), 341 (15,755) nm; IR mmax: 3321, 2912, 2863, 2222, 1634, 1446, 1416, 1385, 1090, 798 cm-1;

EIMS m/z (rel. int.): 230 [M]? (53), 212 (10), 199 (100), 183 (6), 170 (30), 169 (24), 149 (6), 139 (9), 127 (18), 93(9); HREIMS

200 Phytochem Rev (2016) 15:197–220

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Table 1 continued

m/z: 230.0403 (calcd. for C13H10SO2, 230.0401); NMR data (CD3OD, 200 and 128.5 MHz); Roots of Balsamorhiza sagittata

(Pursch) Nuttall (family: Asteraceae) (Matsuura et al. 1996)

8. 10,11-Cis-xanthopappin B; 5-(2-Chloro-1-hydroxyethyl)-2-(Z)-hept-5-ene-1,3-diynylthiophene

Colourless oil; [a]D ?10 (c 0.5, acetone); UV kmax (CH3OH): 315, 252, 268, 213 nm; IR (KBr) cmax: 3382 (OH), 2918 (CH3), 2200

(C:C) cm-1; HRESIMS m/z: 251.0302 [M?H]? (calcd. for C13H11ClOSNa, 251.0294); NMR data (CDCl3, 600 and 150 MHz);

Whole plant of Xanthopappus subacaulis C. Winkl (family: Asteraceae) (Zhang et al. 2014)

9. Xanthopappin B; 5-(2-Chloro-1-hydroxyethyl)-2-(E)-hept-5-ene-1,3- diynylthiophene

Brown oil; [a]D 0 (c 0.377, acetone); UV kmax (CH3OH) (log e): 209 (4.37), 268 (4.44) nm; EIMS m/z (rel. int.): 252 [M?2]? (14),

251 [M?1]? (6), 250 [M]? (38), 201 [M–CH2Cl]? (100), 171 [M-CH2ClCH(OH)]

? (18); HRTOFMS m/z: 273.0112 [M?Na]?

(calcd. for C13H11ONaSCl, 273.0116); NMR data (CDCl3, 500 and 125 MHz); The stems and roots of Xanthopappus subacaulis

C. Winkl (family: Asteraceae) (Tian et al. 2006)

10. 5-(But-4-chloro-3-hydroxy-1-ynyl)-2-(Z)-pent-3-ene-1-ynylthiophene

Colourless oil; [a]D -10 (c 1.0, acetone); UV kmax (CH3OH): 211, 227, 316, 333 nm; IR (KBr) cmax 3344 (OH), 2924 (CH3), 2219

(C:C) cm-1; HRESIMS m/z: 273.0115 [M?Na]? (calcd. for C13H11ClOSNa, 273.0111); NMR data (CDCl3, 600 and

150 MHz); Whole plant of Xanthopappus subacaulis C. Winkl (family: Asteraceae) (Zhang et al. 2014)

11. 5-(But-4-chloro-3-hydroxy-1-ynyl)-2-(E)-pent-3-ene-1-ynylthiophene

Colourless oil; [a]D ?10 (c 1.0, acetone); UV kmax (CH3OH): 210, 226, 319, 334 nm; IR (KBr) cmax: 3344 (OH), 2924 (CH3), 2180

(C:C) cm-1; HRESIMS m/z: 273.0115 [M?Na]? (calcd. for C13H11ClOSNa, 273.0111); NMR data (CDCl3, 600 and

150 MHz); Whole plant of Xanthopappus subacaulis C. Winkl (family: Asteraceae) (Zhang et al. 2014)

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Table 1 continued

12. 5-(1,2-Dihydroxyethyl)-2-(Z)-hept-5-ene-1,3-diynylthiophene

Colourless oil; [a]D ?30 (c 10.0, acetone); UV kmax (CH3OH): 316, 268, 253, 216 nm; IR (KBr) cmax: 3359 (OH), 2921(CH3),

2204 (C:C) cm-1; HRESIMS m/z: 255.0457 [M?Na]? (calcd. for C13H12O2SNa, 255.0450); NMR data (CDCl3, 600 and

150 MHz); Whole plant of Xanthopappus subacaulis C. Winkl (family: Asteraceae) (Zhang et al. 2014)

13. 5-(But-3,4-dihydroxy-1-ynyl)-2-(Z)-pent-3-ene-1-ynylthiophene

Colourless oil; [a]D ?40 (c 1.0, acetone); UV kmax (CH3OH): 312, 261, 213 nm; IR (KBr) cmax: 3363 (OH), 2923 (CH3), 2227

(C:C) cm-1; HRESIMS m/z: 255.0456 [M?Na]? (calcd. for C13H12O2SNa, 255.0450); NMR data (CDCl3, 600 and 150 MHz);

Whole plant of Xanthopappus subacaulis C. Winkl (family: Asteraceae) (Zhang et al. 2014)

14. 5-(But-3,4-dihydroxy-1-ynyl)-2-(E)-pent-3-ene-1-ynylthiophene

Colourless oil; [a]D ?40 (c 1.0, acetone); UV kmax (CH3OH): 313, 263, 2I5 nm; IR (KBr) cmax: 3344 (OH), 2924 (CH3), 2180

(C:C) cm-1; HRESIMS m/z: 255.0456 [M?Na]? (calcd. for C13H12O2SNa, 255.0450); NMR data (CDCl3, 600 and 150 MHz);

Whole plant of Xanthopappus subacaulis C. Winkl (family: Asteraceae) (Zhang et al. 2014)

15. 2-Acetyl-3-metoxy-5-(prop-1-ynyl) thiophen

A white solid crystal; mp 71–73 �C; UV kmax 300 nm; IR mmax: 1545 (Ar), 1632 (CO), 2233 (C:C) cm-1; CIMS m/z (rel. int.): 195

[M?H]? (100); EIMS m/z (rel. int.): 194 [M]? (93.3), 179 [M–CH3]? (100), 165 (22.8), 151 [M–CH3CO]

? (30.5), 136 (20.9),

108 (26.6), 93 (20.5), 77 (21.9), 63 (61), 43 (67); NMR data (CDCl3, 400 and 100 MHz); Roots of Artemisia absinthium L.

(family: Asteraceae) (Yamari et al. 2004)

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Table 1 continued

16. 5-Hydroxymethyl-2-(E)-hept-5-ene-1,3-diynylthiophene diol

Cream crystals; mp 72–75 �C; UV kmax (CH3OH): 217, 227 sh, 256, 261, 271, 304 sh, 321, 348 sh nm; IR (KBr) cmax: 3260, 2900,

210, 2150, 1613, 1440, 1362, 1349, 1285, 1182, 1130, 1030, 995, 942, 805 cm-1; EIMS m/z (rel. int.): 202 [M]? (100) 185 [M–

H2O]? (46), 74 [M-CO]? (16), 171 [M–CH2OH]

? (20); HRTOFMS m/z: 203.0525 [M?H]? (calcd. for C12H11OS, 203.0530);

NMR data (CDCl3, 90 MHz for 1H and 125 for 13C NMR); Roots of Leuzea carthamoides DC (syn. Rhaponticum carthamoides

Willd. Iljin) (family: Asteraceae); (Szendrei et al. 1984; Tian et al. 2006)

17. (E)-2-[5-(Hept-5-en-1,3-diynyl)-thien-2-yl]-ethan-1,2-diol

Cream crystals; mp 96–98 �C; [a]D 0 (c 0.083, acetone); UV kmax (CH3OH): 217, 255, 270, 304 sh, 321, 346 sh nm; IR (KBr) cmax:

3200 (br), 2870, 2160, 2100, 1610, 1435, 1285, 1200, 1160, 1090, 1055, 1040, 940, 875, 810 cm-1; EIMS m/z (rel. int.): 232

[M]? (25), 201 [M-CH2OH]? (l00), 171 [M–CH(OH)–CH2OH]

?; HRTOFMS m/z: 255.0525 [M?Na]? (calcd. for C13H12ONaS,

255.0455); NMR data (CDCl3, 600 and 150 MHz); Underground parts of Leuzea carthamoides DC (syn. Rhaponticum

carthamoides Willd. Iljin) (family: Asteraceae) (Chobot et al. 2003; Szendrei et al. 1984; Tian et al. 2006)

18. 2-[Pent-1,3-diynyl]-5[4-hydroxybut-1-ynyl]-thiophene

Yellowish oil; IR (KBr) cmax: 3490, 2230, 1640, 1100, 980 cm-1; 13C NMR (CDCl3, 75 MHz): dC 135.62, 132.94, 128.12, 123.73,

95.46, 85.35, 75.28, 70.25, 67.98, 65.30, 61.96, 25.43, 4.91; EIMS m/z (rel. int.): 214 (30), 187 (100); 1H NMR data (CDCl3,

90 MHz); Roots of Echinops pappii Chiov (family: Asteraceae) (Abegaz 1991)

19. 2-[Cis-pent-3-en-l-ynyl]-5-[4-hydroxybut-l-ynyl]-thiophene

20. 2-[Trans-pent-3-en-l-ynyl]-5-[4-hydroxybut-l-ynyl]-thiophene

Yellow waxy solid; IR (KBr) cmax: 3360, 3040, 2245, 2165, 1630, 1200, 1058, 960, 820 cm-1; 13C NMR data (CDCl3, 75 MHz):

dC 141.90, 140.60, 132.48, 132.55, 132.33, 132.06, 125.42, 125.10, 122.66, 111.63, 110.39, 92.80, 92.43, 92.31, 91.28, 83.80,

81.07, 77.79, 75.40, 61.17, 61.13, 24.02, 18.79, 16.19; HRMS m/z: 216.0612 (calcd. for C13H12OS; 216.0609); EIMS m/z (rel.

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Table 1 continued

int.): 216 (24), 189 (100); NMR data (CDCl3, 300 and 75 MHz); Roots of Echinops pappii Chiov (family: Asteraceae) (Abegaz

1991)

21. PDDYT; 2-(Penta-1,3-diynyl)-5-(3,4-dihydroxybut-1-ynyl)-thiophene

EIMS m/z: 230 [M]?; NMR data (CD3OD, 500 and 125 MHz); Roots of Echinops grijsii Hance (family: Asteraceae) (Jin et al.

2008; Shi et al. 2010)

22. 4-(5-(Penta-1,3-diynyl)thiophen-2-yl)but-3-ynyl acetate

NMR data (CDCl3, 90 MHz); Roots of Echinops hispidus Fresen (family: Asteraceae) (Abegaz et al. 1991)

23A. 2-Hydroxy-4-(5-(penta-1,3-diynyl)thiophen-2-yl)but-3-ynyl acetate

IR (KBr) mmax: 2237, 1758, 1240 cm-1; HRMS m/z (rel. int.): 272. 0507 (calcd. for C15H12O3S; 272.0510) (24), 254 (10), 212 (100),

199 (42), 170 (20); NMR data (CDCl3, 90 MHz); Roots of Echinops hispidus Fresen (family: Asteraceae) (Abegaz et al. 1991)

23B. 2-(Pant-1,3-diynyl)-5-(4-acetoxy-3-hydroxybuta-1-ynyl)-thiophene

EIMS m/z: 290 [M]?; NMR data (CDCl3, 500 and 125 MHz); Stems and leaves of Pluchea indica (L.) Less. (family: Asteraceae)

(Jin et al. 2008)

24. 1-Hydroxy-4-(5-(penta-1,3-diynyl)thiophen-2-yl)but-3-yn-2-yl acetate

204 Phytochem Rev (2016) 15:197–220

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Table 1 continued

NMR data (CDCl3, 90 MHz); Roots of Echinops hispidus Fresen (family: Asteraceae) (Abegaz et al. 1991)

25. 4-(5-(Penta-1,3-diynyl)thiophen-2-yl)but-3-yne-1,2-diyl diacetate

NMR data (CDCl3, 90 MHz); Roots of Echinops hispidus Fresen (family: Asteraceae) (Abegaz et al. 1991)

26. 2-Chloro-4-(5-(penta-1,3-diynyl)thiophen-2-yl)but-3-yn-1-ol

NMR data (CDCl3, 90 MHz); Roots of Echinops hispidus Fresen (family: Asteraceae) (Abegaz et al. 1991)

27A. 4-[5-(Penta-1,3-diynyl)thien-2-yl]-2-chlorobut-3-ynyl acetate

EIMS m/z (rel. int.): 292 (5), 290 [M]? (17), 254 (28), 230 (100), 195 (59); NMR data (CDCl3, 400 and 100 MHz); Roots of

Echinops transiliensis Golosh (family: Asteraceae) (Fokialakis et al. 2006)

27B. 2-(Pant-1,3-diynyl)-5-(4-acetoxy-3-chlorobuta-1-ynyl)-thiophene

EIMS m/z: 272 [M]?; NMR data (CDCl3, 500 and 125 MHz); Stems and leaves of Pluchea indica (L.) Less. (family: Asteraceae)

(Jin et al. 2008)

28. 5-(1,2-Diacetoxyethyl)-2-(E)-hept-5-ene-1,3-diynylthiophene diol

Yellow oil; UV kmax (CH3OH): 217, 256, 261, 270, 302 sh, 322, 344 sh nm; IR (KBr) cmax: 2960, 2200, 2140, 1755, 1422, 1370,

1226, 1049, 950, 870, 810 cm-1; EIMS m/z (rel. int.): 316 [M]? (41), (256) [M–AcOH]? (55), 214 [M–AcOH–CH,CO]? (l00),

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1977). Oleic acid has been proposed as a precursor in

the biosynthesis of thiophenes via acetylene interme-

diates (Margl et al. 2001). Acetylenic natural products

include all compounds containing a carbon–carbon

triple bond or alkynyl functional group. Three fatty

acids have been identified as the basic building blocks

of most acetylenic natural products, including

crepenynic acid, stearolic acid and tariric acid (Minto

and Blacklock 2008). Oleic acid is converted to

trideca-3,5,7,9,11-pentayn-l-ene (PYE) via repeated

desaturation steps involving crepenynic acid and chain

shortening processes (Margl et al. 2001). PYE is then

converted to a variety of different thiophenes that

subsequently accumulate in plant tissue (Fig. 2)

(Jacobs et al. 1995). The biosynthesis of polyacetyle-

nes occurs in two stages, including (A) an oxidative

dehydrogenation (desaturation) mechanism, where the

existing alkene functionality undergoes a desaturation

reaction through an iron-catalyzed dehydrogenation

with molecular oxygen. The electrons required by this

reaction are provided by either NADH or NADPH.

The second step (B) involves a decarboxylative enol

elimination mechanism, which uses a divergent

approach for the formation of the second p-bond(Fig. 3). The elimination of an activated enol car-

boxylate intermediate is thermodynamically driven by

the formation of CO2, which could be accompanied by

the hydrolysis of the pyrophosphate. According to the

original hypotheses, path A would operate with full-

length acyl lipids, whereas path B would install

acetylenic groups during de novo fatty acid biosyn-

thesis. Although the current paradigm and all ex-

periments dealing with fatty acid biosynthesis are

consistent with the desaturase pathway, the elimina-

tion hypothesis remains valid for polyketide-derived

acetylenic natural products (Minto and Blacklock

2008).

Sulfur, which is a heteroatom commonly intro-

duced into polyacetylenes, is found in a wide range of

ecologically significant thiophenes and bithiophenes.

The structures of these compounds vary considerably

in terms of their number of thiophene rings (1–3) and

the degree of unsaturation in their side chains (i.e.,

ene/yne) (Margl et al. 2001). Cysteine and H2S have

both been proposed as potential sources of sulfur

(Bohlmann et al. 1973, 1988; Jente et al. 1988, 1981).

The key step in the conversion of PYE to thiophenes is

the addition of H2S or its biochemical equivalent to a

conjugated triple bond, followed by a ring formation

reaction, which is probably a two-step reaction

(Bohlmann et al. 1973). In addition to the formation

of compounds containing two or three thiophene rings,

the removal of a terminal methyl group and modifi-

cation of a vinyl group are necessary to obtain the

various thiophenes that ultimately accumulate in plant

tissues (Fig. 4).

The addition of sulfur to a diyne unit leads to the

formation of a thiophene ring via a stepwise process.

The formal addition of H2S produces vinyl thiols that

are intercepted in certain Asteraceae species to

produce thioethers. Subsequent ring closure results

in the formation of thiophenes and the oxidative

formation of disulfide linkages that producing bithio-

phenes (Fig. 4).

The proportion of thiophenes found in the different

parts of a plant can vary considerably based on the type

Table 1 continued

201 [M–Me–CH2CO]? (82), 171 [M–CH(COOMe)–CH2COOMe]? (9); HRTOFMS m/z: 239.0663 [M?Na]? (calcd. for

C17H16O4NaS, 239.0667); NMR data (CDCl3, 90 MHz for 1H and 125 for 13C NMR); Roots of Leuzea carthamoides DC (syn.

Rhaponticum carthamoides Willd. Iljin) (family: Asteraceae) (Szendrei et al. 1984; Tian et al. 2006)

29. 5-(1-Dihydroxy-2-acetoxyethyl)-2-(E)-hept-5-ene-1,3-diynylthiophene diol

Cream crystals; mp 82–84 �C; UV kmax (CH3OH): 217, 226 sh, 257, 270, 303 sh, 324, 346 sh nm; IR (KBr) cmax: 3310, 2940, 885,

2170, 2210, 1700, 1430, 1385, 1360, 1270, 1235, 1225, 1185, 145, 1080, 1035, 980, 944, 895, 802 cm-1; MS m/z (rel. int.): 274

[M]? (15), 256 [M–H2O]? (2), 214 [M–HOAc]? (70), 201 [M–CH2COOMe]? (l00), 185 [M–C7H5]

? (12), 171 [M–CH(OH)–

CH2COOMe]? (28); NMR data (CDCl3, 90 MHz); Roots of Leuzea carthamoides DC (syn. Rhaponticum carthamoides Willd.

Iljin) (family: Asteraceae) (Szendrei et al. 1984)

206 Phytochem Rev (2016) 15:197–220

123

Table 2 Naturally occurring thiophene: group-II: bithiophene

30. 5-Acetyl-2,20-bithiophene

mp 59–60 �C; UV kmax (CH3OH): 254, 365 nm; NMR data (CDCl3, 500 and 125 MHz); Roots of Echinops latifolius Tausch.

(family: Asteraceae) (Wang et al. 2008)

31. 5-(4-Hydroxybut-1-ynyl)-2,20-bithiophene

UV kmax (CH3OH): 254, 365 nm; NMR data (CDCl3, 500 and 125 MHz); Roots of Echinops latifolius Tausch. (family:

Asteraceae) (Wang et al. 2008)

32. BBT; 5-(But-3-en-1-ynyl)-2,20-bithiophene

UV kmax (CH3OH): 254, 365 nm; NMR data (CDCl3, 500 and 125 MHz); Roots of Echinops latifolius Tausch. (family:

Asteraceae) (Margl et al. 2001; Wang et al. 2008)

33. 5-(3-Acetoxy -4-isovaleroyloxybut-1-ynyl-2,20-bithiophene

mp 94–95 �C; UV kmax (CH3OH): 254, 365 nm; NMR data (CDCl3, 500 and 125 MHz); Roots of Echinops latifolius Tausch.

(family: Asteraceae) (Wang et al. 2008)

34. 5-(3-Hydroxmethyl-3-isovaleroyloxyprop-1-ynyl)-2,20-bithiophene

Phytochem Rev (2016) 15:197–220 207

123

Table 2 continued

Yellow oil; [a]D -9.0 (c 0.001, CHCl3); HRMS: m/z 334.0696 (calcd. for C17H18O3S2, 334.0671); ESIMS m/z (rel. int.): 357.0

[M?Na]? (9.0), 358.0 [M?H?Na]? (1.7), 359.0 [M?2H?Na]? (1.1), 360.2 [M?3H?Na]? (0.3), 254.8 [M?Na-102]?

(100.0); NMR data (CDCl3, 300 and 75 MHz); Roots of Echinops latifolius Tausch. (family: Asteraceae) (Wang et al. 2006)

35. Grijisone A: 5-[(4-Isovaleroyloxy) buta-1-onyl]-2,20-bithiophene

Yellow powder (CDCl3); mp 62.3-62.7 �C; UV (MeOH) kmax (log e): 375 (4.3), 262 (3.2), 220 (3.6) nm; IR (KBr) mmax: 1729,1655, 839, 801, 717 cm-1; HREIMS m/z: 336.0838 [M]? (calcd. for C17H20O3S2, 336.0854); EIMS m/z (rel. int.): 336.1 (100),

337.1 (19.8), 338.0 (10.4), 166 (4.3); NMR data (CDCl3, 600 and 150 MHz); Roots of Echinops grijissi Hance (family:

Asteraceae) (Zhang et al. 2008)

36. 5-(3,4-Diacetoxy-l-butynyl)-2,20-bithiophene

Yellow oil; EIMS m/z (rel. int.): 334 [M]? (25); 274 [M–AcOH]? (l), 232 (49, 95 (4), 73 (5), 43 (100); C16H14O4S2; NMR data

(CDCl3, 200 MHz); Roots of Tagetes patula L. (family: Asteraceae) (Menelaou et al. 1991)

37. 50-Methyl-[5-(4-acetoxy-1-butynyl)]-2,20-bithiophene

Yellow oil; ESIMS m/z (rel. int.): 291 [M?H]? (10), 301 (26), 245 (33), 229 (73), 313 [M?Na]? (100); NMR data (CDCl3, 300

and 75 MHz); Aerial parts of Porophyllum ruderale (Jacq.) (family: Asteraceae) (Takahashi et al. 2013, 2011)

38. Methyl-5-[4-(3-methyl-1-oxobutoxy)-1-butynyl]-2,20-bithiophene

Yellow needle-like crystals; UV kmax (Et2O): 347.2 nm; EIMS m/z (rel. int. %): 245.7 [M]? (94.6), 228.8 (70), 216.8 (100);

HREIMS m/z: 246.0155 (C13H10OS2); NMR (CDCl3, 300 and 75 MHz); Roots of Tagetes patula L. (family: Asteraceae) (Bano

et al. 2002)

208 Phytochem Rev (2016) 15:197–220

123

Table 2 continued

39. Methyl-5-[4-(3-methyl-1-oxobutoxy)-1-butynyl]-2,20-bithiophene

Yellowish oil; UV kmax (Et2O): 339 nm (e 26,726); IR (CCl4) mmax: 2850 (C–H stretching), 2300 (C:C), 1717 (ester carbonyl),

1600 (C=C) cm-1; HREIMS m/z (rel. int.): 332.0903 M]? (C18H20O2S2) (19), 230.0186 [M–C5H10O2]? (100), 217 (9), 197 (5),

115 (7), 102 (4); NMR (CDCl3, 300 and 75 MHz); Roots of Tagetes patula L. (family: Asteraceae) (Bano et al. 2002)

40. Grijisyne A; 5-[2-[4-(5-Propyneylthiophen-2-yl)buta-1,3-diynyl]cyclobutaneyl]ethynyl]-2,20-bithiophene

Yellow powder; mp 134.2–135.0 �C; UV (MeOH) kmax (log e): 340.4 (4.2), 250.6 (3.5) nm; IR (KBr) mmax: 2192, 796, 836,674 cm-1; HREIMS m/z: 412.0429 [M]? (calcd. for C25H16S3, 412.0414); EIMS m/z: 413.0 (100), 414.0 (28.5), 415.1 (12.9),

166 (5.6); NMR data (CDCl3, 600 and 150 MHz); Roots of Echinops grijissi Hance (family: Asteraceae) (Zhang et al. 2008)

41. Cardopatine

Yellow plates; mp 123-125 �C; UV (MeOH) kmax (log e): 340.0 (4.82), 242.0 (4.33) nm; [a]D; IR (KBr) mmax: 840 (2-thienyl), 810(thiophen-2,5-diyl) cm-1; EIMS: m/z (rel. int.): 432 (9) 216.0 (100), 171 (13), 95 (6); NMR data (CDCl3, 400 and 100 MHz);

Stem and leaves of Echinops latifolius Tausch. (family: Asteraceae) (Selva et al. 1978; Zhang et al. 2007)

42. Isodopatine

Light yellow plates; mp 79–80 �C; UV (MeOH) kmax (log e): 340.0 (4.82), 242.0 (4.33) nm; IR (KBr) mmax: 840 (2-thienyl), 810

(thiophen-2,5-diyl) cm-1; EIMS: m/z (rel. int.): 432 (12), 216.0 (100), 171 (13), 95 (7); NMR data (CDCl3, 300 and 75 MHz);

Stem and leaves of Echinops latifolius Tausch. (family: Asteraceae) (Selva et al. 1978; Zhang et al. 2007)

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123

of plant. For example, no thiophenes can be found in the

shoots of achenes, with bithienyls and traces of 5-(but-

3-en-1-ynyl)-2,20-bithiophene (BBT) being identified

as the major chemicals in this case. a-Terthienyl, whichcan be found in the root of corresponding plants but not

in the shoots, and accumulates in flowers. Despite many

experiments, it remains to be shown whether thiophene

metabolites originate exclusively in the roots, and that

specific thiophenes are preferentially accumulated in

the different parts of the plant, or whether enzymatic

components of the thiophene pathway are expressed in a

tissue-specific manner. It has been reported that methyl

cleavage occurs prior to the formation of the second

thiophene ring (Minto and Blacklock 2008).

Methods for separation of thiophenes

Thiophenes extraction and purification

To allow for the exclusive extraction and isolation of

only thiophene-containing compounds, the plant ma-

terials were extracted with a 1:1 (v/v) mixture of EtOH

andH2O. The resulting thiopheneswere then separated

by partitioning them between a 1:1 (v/v) mixture of n-

hexane and tert-butylmethylether (Jacobs et al. 1995).

The individual layers were collected and the organic

solventswere evaporated under a stream ofN2 gas. The

resulting mixture of thiophenes mixture was then

dissolved in EtOH and purified by preparative HPLC

over an octadecylsilane (C18) reversed-phase column,

using 72–85 % acetonitrile or 70–85 % MeOH as an

eluent (Downum et al. 1984). The compounds eluted

from the column were detected using a UV spec-

trophotometer with a detection range of 320–350 nm

(Jin et al. 2008; Norton et al. 1985; Tosi et al. 1991).

Normal phase HPLC analyses were conducted using a

95:5 (v/v) mixture of n-hexane and dioxane as the

eluent (Szarka et al. 2006, 2007). HPLC was used to

identify and quantify the different thiophene-contain-

ing compounds (Camm et al. 1975; Croes et al. 1989).

Thiophenes can generally be isolated by the extrac-

tion of plant materials with EtOH or MeOH, and the

resulting thiophenes can then be further purified by

partitioning the alcohol extract between n-hexane or pet

ether (PE). The n-hexane or PE fraction can then be

subjected to purification by column chromatography

using n-hexane:EtOAc or PE:acetone as the eluent with

a gradient elution system. The isolated compounds can

then be further purified by preparative HPLC.

Another method for the isolation and purification of

thiophenes is the direct extraction of plant materials

with n-hexane or PE. The resulting extracts can be

purified by column chromatography over silica gel

eluting with an n-hexane:EtOAc or PE:acetone gradi-

ent, followed by preparative HPLC.

Table 2 continued

43. Xanthopappin C; 1,2-Bis[5-(E)-hept-5-ene-1,3-diynylthiophen-2-yl]-2-hydroxypentane-1,4-dione

Brown oil; [a]D 0 (c 0.085, acetone); UV kmax (CH3OH) (log e): 215 (4.72), 269 (4.83), 353 (4.52) nm; EIMS m/z: 456 [M]? (4),

398 [M–CH3COCH3]? (17), 370 [M–CH3COCH3–H2O]

? (9), 257 [M–HCOC4H2SC:CC:CCH=CHCH3]? (25), 200

[HCOC4H2SC:CC:CCH=CHCH3]? (18), 199 [HCOC4H2SC:CC:CCH=CHCH3–H]

? (100), 171 (6); HRTOFMS m/z:

479.0737 [M?Na]?(calcd. for C27H20O3NaS2, 479.0751); NMR data (CDCl3, 500 and 125 MHz); The stems and roots of

Xanthopappus subacaulis C. Winkl (family: Asteraceae) (Tian et al. 2006)

210 Phytochem Rev (2016) 15:197–220

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Table 3 Naturally occurring thiophene: group-III: terthiophene

44. a-Terthiophene

Colourless needles; mp 91–92 �C; IR (KBr) mmax: 3434, 2931, 2862, 1637, 1460, 1378, 1240, 1050, 1021, 969, 958, 837,

800 cm-1; C12H8S3; NMR data (Acetone-d6, 500 and 125 MHz); Roots of Echinops grijissii Hance (family: Asteraceae) (Liu

et al. 2002)

45. 5-Acetyl-a-terthiophene

Yellow crystals; mp 135–1378 �C; C14H10S3O; IR (KBr) mmax: 2919, 2850, 1731, 1636 cm-1; EIMS m/z: 290 [M]?, 275 [M–

CH3]?, 247 [M–CH3CO]

?, 203, 138; NMR data (Acetone-d6, 500 and 125 MHz); Roots of Echinops grijisii Hance (family:

Asteraceae) (Liu et al. 2002)

46. 5-Chloro-a-terthiophene

Yellow crystals; mp 129–130 �C; C12H7S3Cl; IR (KBr) mmax: 2914, 1586, 1422, 833, 788, 686 cm-1; EIMS m/z: 284 [M?2]?,

282 [M]?, 247 [M–Cl]?, 237, 214, 203, 141, 127, 102, 93; NMR data (Acetone-d6, 500 and 125 MHz); Roots of Echinops

grijisii Hance (family: Asteraceae) (Liu et al. 2002)

47. 5,50 0-Dichloro-a-terthiophene

Yellow crystals; mp 134–135 �C; C12H6S3Cl2; IR (KBr) mmax: 2914, 1427, 849, 787 cm-1; EIMS m/z: 320 [M?4]?, 318 [M?2]?,

316 [M]?, 281 [M–Cl]?, 246 [M–2Cl]?, 237, 201, 158, 145, 119; NMR data (Acetone-d6, 500 and 125 MHz); Roots of

Echinops grijisii Hance (family: Asteraceae) (Liu et al. 2002)

48. 5-Methyl-2,20:50,20 0-terthiophene

Viscous yellow oil; ESIMS m/z (rel. int.): 185 (15), 229 (25), 263 [M?H]? (36), 262 (100); NMR data (CDCl3, 300 and 75 MHz);

Aerial parts of Porophyllum ruderale (family: Asteraceae) (Takahashi et al. 2013, 2011)

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123

TLC chromatography and detection of thiophenes

The following solvent systems were used for TLC

analysis: PE:acetone (99:1), PE, PE:diethyl ether

(90:10), and n-hexane:dioxane:n-BuOH (75:25:1)

(Margl et al. 2001). Thiophenes can be detected on a

TLC plate by their characteristic fluorescence under

long wave UV light or by their reaction with one of the

following TLC stains:

1. Vanillin spray reagent (0.5 g vanillin ? 9 mL

95 % EtOH ? 0.5 mL conc. H2SO4 ? 3 drops

glacial acetic acid (Picman et al. 1980).

2. Isatin spray reagent (0.4 % isatin in conc. H2SO4)

(Curtis and Phillips 1962).

Structural elucidation of the thiophenes

Nuclear magnetic resonance spectroscopy

Nuclear magnetic resonance spectroscopy (NMR) is

one the most powerful techniques available for

investigating the structural properties of different

molecules. One of the main applications of NMR in

thiophene research is the structural elucidation of

novel compounds based on their 1D (1H, 13C and

DEPT) and 2D (1H–1H COSY, HSQC/HMQC and

HMBC) NMR data (Tables 1, 2, 3, 4, 5). The

connectivities of the different atoms present in the

thiophenes isolated in the current study were estab-

lished by NOE and ROESY experiments to determine

the stereochemistries of the different thiophenes.

Mass spectroscopy (MS)

Mass spectroscopy has been used as an effective

method for the identification and quantitative deter-

mination of thiophenes. The mass spectra of sulfur-

containing compounds generally contain a series of

characteristic fragments, including [M]?, [M?H]?

and [M?2H]? (corresponding to 4.5 % of the inten-

sity of theM?�ion). Electrospray ionization (ESI) mass

spectrometry generally gives [M?H]? and [M?Na]±

ions for sulfur-containing compounds. It is noteworthy

that sulfur can be lost from the M?� ions of sulfur-

containing compounds together with neighboring C

atoms as CHS fragments. These fragments would

appear with m/z values of 45 (CHS?) and 44 (CS?�),

and can be used as indicators for the presence of sulfur

(Pretsch et al. 2009).

Table 3 continued

49. Ecliptal; 5-Formyl-a-terthiophene

mp 144–145 �C; EIMS m/z: 276 [M]?; NMR data (CDCl3, 300 and 75 MHz); Herbs of Eclipta alba Hassk (family: Asteraceae)

(Das and Chakravarty 1991; Yuan et al. 2007)

Table 4 Naturally occurring thiophene: group-IV: quinquethiophene

50. 5-Methyl-2, 20,50, 20 0,50 0,20 0 0,50 0 0,20 0 0-quinquethiophene

Brown needles; mp 215–216 �C; UV (MeOH) kmax: 334, 387 nm; IR (KBr) mmax: 2870, 1600 cm-1; HRESIMS m/z: 427.6611

[M?H]? (calcd. for C21H15S5, 427.6609); 428.6613 [M?2H]? (calcd. for C21H16S5, 428.6609); NMR data (CDCl3, 500 and

125 MHz); Leaves of Tagetes minuta L. (family Asteraceae) (Al-Musayeib et al. 2014)

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Table 5 Naturally occurring thiophene: group-V: miscellaneous

51. Echinothiophenegenol; 5-Hydroxy-6-[(1E,3E)-6-hydroxy-1,3-hexadienyl]-2-hydroxymethyl-thieno[2,3-e]-isobenzofuran-8(6H)-one

Pale yellow powder; IR (KBr) mmax: 3436, 1697, 1467 cm-1; ESIMS m/z: 332 [M?H]?; HRESIMS m/z: 331.0643 [M–H]-

(calcd. 331.0640); NMR data (DMSO-d6, 600 and 150 MHz); Roots of Echinops grijissii Hance (family: Asteraceae) (Zhanga

et al. 2009)

52. Echinothiophene; 5-O-b-D-glucopyranosyl-6-[(1E,3E)-6-hydroxy-1,3-hexadienyl]-2-hydroxymethyl-thieno[2,3-e]-isobenzofuran-8(6H)-one

Phytochem Rev (2016) 15:197–220 213

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Biological activity

Despite the unique nature of their chemical structures

relative to the many other different classes of naturally

occurring compounds, thiophenes have not yet been

well studied in terms of their potential pharmaco-

logical activities. Several naturally occurring thio-

phenes and thiophene-rich extracts have exhibited a

variety of different biological effects, including

antimicrobial, cytotoxic, chemo-preventive, photo-

toxic, insecticidal, herbicidal, and anti-leishmanial

activities.

Antimicrobial activities

Some of the thiophenes isolated in the current study

exhibited antibacterial, antifungal and antiviral ac-

tivities towards a variety of different microorganisms.

Saha et al. (2013) reported that the isolation of a

thiophene-rich extract from Tagetes minuta exhibited

moderate antifungal activity towards several soil

borne and foliar plant pathogens, including

Rhizoctonia solani, Sclerotinia sclerotiorum, and

Sclertium rolfsii. These results therefore indicated

that Tagetes minuta could be used as a potential

candidate for the production of natural fungicides.

Furthermore, the methanol extract of Tagetes patula

exhibited a dose dependent anti-fungal activity to-

wards several phytopathogenic fungi, including Botry-

tis cinerea, Fusarium moniliforme, and Pythium

ultimum. It is noteworthy that the methanol extract

of Tagetes patula exhibited much stronger antifungal

activity when it was used in light than it did in the dark.

The enhanced antifungal activity of the extract in the

presence of light could be attributed to light-induced

changes in the fungal cell membranes involving the

production of free radicals, which could result in the

premature aging of the fungal mycelia (Mares et al.

2004).

Compound 3, which was isolated from Xanthopap-

pus subcaulis, exhibited potent antibacterial activity

against Bacillus subtilis with an MIC of 7.25 lg/mL.

In contrast, the corresponding erythro isomer 4

exhibited broad spectrum antibacterial activity

Table 5 continued

Pale-yellowish needles; mp 214–216 �C (dec); IR (KBr) mmax: 3421, 2924, 1750, 1645, 1094, 1056 cm-1; UV kmax (MeOH) (log

e): 254 (4.94), 319 (4.10) nm; HRFABMS m/z: 495.1339 [M?H]? (D0.0022 of the calcd.) and m/z: 517.1147 [M?Na]?

(D0.0010 of the calcd.) (C23H26O10S); NMR data (DMSO-d6, 500 and 125 MHz); Roots of Echinops grijissii Hance (family:

Asteraceae) (Koike et al. 1999)

214 Phytochem Rev (2016) 15:197–220

123

towards Escherichia coli, B. cereus, Staphylococcus

aureus, and Erwinia carotovora with MIC values of

12.5, 15.5, 7.2, and 7.2 lg/mL, respectively. Several

other chlorinated derivatives (8–11), which were

isolated from the same plant, exhibited only moderate

activity towards E. coli, B. cereus, S. aureus, E.

carotovora, and B. subtilis (Zhang et al. 2014).

Compound 5, which was isolated from the chloroform

extract of Chrysanthemum coronarium, exhibited

moderate antimicrobial activity towards B. subtilis,

Pseudomonus aeruginosa, Candida albicans and

Trichophyton mentagrophytes (Ragasa et al. 1997).

Compound 7 was isolated from Balsamorhiza sagit-

tata, which is a plant native to Northwestern America.

This plant has been reported as a folk medicine

because of its antibacterial and antifungal activities,

and compound 7 exhibited significant activities again-

st B. subtilis, S. aureus and S. aureus SA0017, which is

a methicillin-resistant strain of S. aureus. The an-

tibacterial activity of compound 7 towards a variety of

different bacteria was enhanced when the experiments

were conducted in the presence of UV-A light

Fig. 2 Biosynthesis of

different thiophenes

Phytochem Rev (2016) 15:197–220 215

123

(Matsuura et al. 1996). Furthermore, the antibacterial

activity of compound 7 was confirmed by Kundu and

Chatterjee (2013), who reported that this compound

exhibited MIC values in the range of 25–100 lg/mL

towards six different strains of S. aureus. The authors

of this study also conducted a series of mechanistic

studies with compound 7, which revealed that this

compound exhibited bacteriostatic effects. Further-

more, compound 7 was determined to be a DNA

polymerase inhibitor, as confirmed by agarose gel

electrophoresis (Kundu and Chatterjee 2013). Com-

pound 17 was isolated from Leuzea carthamoides, and

exhibited significant broad spectrum antifungal ac-

tivity towards a variety of different fungal strains, with

Trichophyton mentagrophytes var. mentagrophytes,

Absidia corymbifera and Candida tropicalis being

particularly sensitive to this compound (Chobot et al.

2003).

Thiophenes 18, 27, 31, 32, 36, 40 and 44 were

identified in the dichloromethane extract of Echinops

ritro using an antifungal/biological activity guided

approach. Compounds 18, 31 and 44 exhibited the

most potent antifungal activities of the seven different

compounds towards a variety of different plant

A BFig. 3 The two distinct

proposals for the biogenesis

of acetylenic bonds

Fig. 4 Sulfur addition to

polyacetylenes

216 Phytochem Rev (2016) 15:197–220

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pathogens, including Colletotrichum acutatum, Col-

letotrichum fragrariae and Colletotrichum gloeospo-

rioides at concentrations in the range of 3–30 lM.

Compound 44 appeared to be relatively selective

towards Colletotrichum species, exhibiting a high

level of activity against C. gloeosporioides (IC50\1.6 lM), whilst showing only moderate levels of

activity towards C. acutatum and C. fragariae with

IC50 values of 3.0 and 4.9 lM, respectively. Com-

pound 18 appeared to demonstrate selective antifungal

activity towards Phomopsis species, with moderate

activities towards Phomopsis obscurans (IC50 = 2.9 -

lM) and Phomopsis viticola (IC50\ 1.6 lM). Fur-

thermore, compound 18 exhibited a high level of

activity towards Fusarium oxysporum with an IC50 of

9.5 lM. The high activity of compound 18 is

particularly interesting because very few chemicals

have been reported to inhibit the activity of F.

oxysporum with IC50 values of\30 lM (Fokialakis

et al. 2006).

Compound 49 showed promising inhibitory activity

towards HIV-1 protease with an IC50 value of 58 lM,

but did not show any activity towards HIV-1 integrase

(Tewtrakul et al. 2007). It is noteworthy that com-

pound 44 exhibited a dose-dependent inhibitory

activity towards HIV in the presence of UV-A light

(320–400 nm), but no activity in the presence of

visible light or in the dark. However, compound 44 did

not exhibit any activity towards poliovirus or cox-

sackievirus (Hudson et al. 1993).

Compounds 44, 48, and 49 exhibited photo-induced

inhibitory activity towards the growth of S. aureus. It

is noteworthy that the unsubstituted 2,20:50,200-terthio-phene (44) was the only one of these three compounds

to exhibit inhibitory activity towards E. coli with an

MIC value of 0.62 lg/mL. Furthermore, none of these

three compounds exhibited inhibitory activity towards

P. aeruginosa (Ciofalo et al. 1996).

Antiparasitic activity

Compounds 37 and 48 exhibited antileishmanial

activity towards the promastigote and axenic forms

of Leishmania amazonensis with IC50 values of 7.7

and 21.3 lg/mL, and 19.0 and 28.7 lg/mL, respec-

tively (Takahashi et al. 2011). Both of these com-

pounds were shown to be highly selective towards

intracellular amastigotes with minimal toxicity to-

wards human cells. Furthermore, changes in the

mitochondrial membrane were observed in promastig-

otes treated with compound 37, as well variations in

the morphological characteristics of the cells (Taka-

hashi et al. 2013).

Compound 44 also exhibited significant nematici-

dal activity when it was irradiated with near UV light.

The nematicidal activity of this compound was

attributed to the liberation of reactive oxygen species

from the compound upon UV irradiation (Bakker et al.

1979).

Phototoxic, insecticidal, and herbicidal effects

There is a growing interest in the discovery of

phototoxic phytochemicals, especially those charac-

terized by significant increases in their activities

following exposure to light. These compounds are

mainly used as insecticides, herbicides and antimicro-

bial agents. Thiophenes are a class of natural products

that have been extensively studied in terms of their

phototoxic effects.

The herbicidal activity of a-terthienyl (44) was

assessed in pot and field trials by Lambert et al. (1991).

The results of this study revealed that compound 44

acted as a contact herbicide in corn and broad leaf

weeds with IC50 values in the range of 15–29 kg ha-1.

Compounds 2, 9, 16, 17, 28 and 43 showed photo-

activated insecticidal activity towards the fourth

instar-larvae of the Asian tiger mosquito with LC50

values of 0.71, 0.53, 0.30, 4.2, 0.66, and 0.95 lg/mL,

respectively. In the absence of light, the LC50 values of

compounds 2, 16, 17, 28 and 43 were [10 lg/mL,

while that of compound 9 was 5.1 lg/mL. These

results demonstrated that the irradiation of compounds

2, 16, 17, 28 and 43with light led to 14.1-, 15.2-, 10.5-,

33.3- and 2.4-fold increases in their activity, respec-

tively, as well as a 9.6-fold increase in the activity of 9.

The photo-activated insecticidal effects of these

compounds were attributed to light dependent toxicity

mechanisms involving the photo-oxidation of insect

targets resulting in membrane damage, enzyme inac-

tivation, cell death and other biological loss of

function mechanisms (Tian et al. 2006).

Compound 17was isolated from the roots of Leuzea

carthamoides and exhibited potent phototoxic effects

in histidine photo-oxidation, Artemia salina and

Tubifex assays compared with the known phototoxic

agent xanthotoxin. The higher activity of 17 towards

A. salina could be attributed to the release of singlet

Phytochem Rev (2016) 15:197–220 217

123

oxygen from 17 following its irradiation with light

rather than the release of a superoxide anion, as is the

case with xanthotoxin. A. salina is much more

sensitive to singlet oxygen than it is to superoxide

anion radicals, which explains the higher activity of 17

towards A. salina compared with xanthotoxin (Chobot

et al. 2003).

Cytotoxic effect

Several thiophenes were screened to determine their

cytotoxic effects against a wide range of human cancer

cell lines. The marine sponge-derived thiophene 1

exhibited weak cytotoxicity towards Vero cells

(African green monkey kidney cells) with an IC50

value of 31 lM (Pedpradab and Suwanborirux 2011).

Compounds 35 and 40, which were isolated from the

roots of Echinops grijisii, were evaluated in terms of

their cytotoxic activity towards a variety of different

cancer cell lines, including HL-60, K562 and MCF-7

cells. Compound 35 exhibited moderate activities

against HL60 and K562 cells, with IC50 values of

21.1 and 25.2 lg/mL, respectively. Compound 40 also

exhibited moderate levels of activity against HL60,

K562, and MCF-7 cells, with IC50 values of 19.6, 18.9

and 28.7 lg/mL (Zhang et al. 2008). Compounds 25,

32, 33, 36, 40, and 43were also isolated fromEchinops

grijisii and screened for their cytotoxic activity against

HepG2, K562, HL60, and MCF-7 cells. Compound 36

exhibit a high level of activity towards HL60 andK562

cells with IC50 values of 12 lg/mL), while 33 showed

potent activity towards K562 cells (IC50 = 7 lg/mL).

Jin et al. (2008) reported that most thiophenes are

cytotoxic after UV irradiation. The UV light-mediated

cytotoxicity of thiophenes has been attributed to them

being highly conjugated and becoming increasingly

unstable under UV irradiation conditions. The irra-

diation of these compounds with UV light would

therefore result in the liberation of free radicals that

would attack the cells. However, the main interest of

the authors of this particular study was the structure

activity relationships of compounds that exhibited

cytotoxic activity in the absence of light. The authors

reported that the introduction of an acyl substituent as a

side chain was essential to the cytotoxic activity of

these compounds, especially in the non-radiated

bithiophenes.

Thiophenes have also been reported to exhibit a

variety of other activities, including antimutagenic

and chemopreventive effects. Compound 5 was

investigated in terms of its antimutagenic effects

using a micronucleus test. At a dose of 8 mg/kg bwt,

compound 5 reduced the number of micronucleated

polychromatic erythrocytes by 66.5 % (Ragasa et al.

1997). Compound 21 was reported to possess potent

NAD(P)H: quinine oxidoreductase 1 (NQO1) induc-

ing activity in murine Hepa1c1c7 cells. The maximum

induction of this compound was 3.3-fold greater than

that of 40-bromoflavone (positive control) at a con-

centration of 40 lM. As a phase 2 detoxifying enzyme

inducer, the mechanism of action of compound 21was

investigated to determine whether it was monofunc-

tional (i.e., progressing through the Keap1-Nrf2

pathway) or bifunctional (i.e., progressing through

the aryl hydrocarbon receptor-xenobiotic response

element pathway). The study concluded that com-

pound 21 was acting in a mono-functional manner

though the activation of the Keap1-Nrf2 pathway (Shi

et al. 2010).

Conclusions

Thiophenes are a class of heterocyclic aromatic

compounds that fulfill all the requirements for being

lead compounds in a number of different therapeutic

areas. Compounds belonging to this class possess a

variety of different chemical compositions, and have

been reported to exhibit a wide range of biological

activities. In this review, we have described the

biosynthetic pathways, spectral data, sources and

biological activities of 52 different thiophenes.

Conflict of interest The authors declare that they have no

conflicts of interest.

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