Chinese Chemical Letters  2016, Vol. 27 Issue (5): 643-648   PDF    
Antiviral stereoisomers of 3,5-bis(2-hydroxybut-3-en-1-yl)-1,2,4-thiadiazole from the roots of Isatis indigotica
Ming-Hua Chena,b, Sheng Lina, Ya-Nan Wanga, Cheng-Gen Zhua, Yu-Huan Lib, Jian-Dong Jianga,b, Jian-Gong Shia     
a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China ;
b Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Abstract: Four stereoisomers of 3,5-bis(2-hydroxybut-3-en-1-yl)-1,2,4-thiadiazole, named insatindigothiadiazoles A-D (1a-1d), were isolated from the roots of Isatis indigotica. Their structures were determined by spectroscopic analysis; specifically, the absolute configurations were assigned by using the MPA determination rule based on ΔδRS values of MPA esters, and supported by electronic CD (ECD) calculations. Proposed biosynthetic pathways and preliminary investigations of the biological activities of 1a-1d against influenza virus A (H3N2), Coxsackie virus B3, and/or HSV-1 are also discussed.
Key words: Isatis indigotica     Cruciferae     3,5-Bis(2-hydroxybut-3-en-1-yl)-1,2,4-thiadiazole     Stereoisomer     Insatindigothiadiazoles A-D     Antiviral activity    
1. Introduction

Isatis indigotica Fort.,a biennial herbaceous plant of the Cruciferae family,is widely cultivated to meet demands of medicinal utilization in China. Its dried roots and leaves,named "ban lan gen" and "da qing ye" in Chinese,respectively,are commonly used in traditional Chinese medicine for the treatment of influenza,cold,fever,and other infections [1]. Many formulations containing "ban lan gen" and/or "da qing ye" are marketed and recorded in Chinese Pharmacopoeia [2],which play an important role to treat and prevent influenza during influenza pandemics in China. Clinical efficacy,together with diverse structures and biological activities from extracts of these herbal medicines,has long attracted interest from chemists and pharmacologists. Pharmacological studies showed that extracts of these medicines displayed antiviral,antiendotoxic,antinociceptive, antiinflammatory,and antipyretic effects and cytotoxicity against leukemia cells [3, 4, 5, 6, 7]. Meanwhile,bioactive chemical components,such as alkaloids,lignans,ceramides,flavonoids, and sulfur containing metabolites [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19],were isolated from the extracts. However,previous chemical studies were mainly performed on the ethanol and methanol extracts of the drug materials,which is inconsistent with their practical application by decocting with water. Therefore,as part of a program to assess the chemical and biological diversity of traditional Chinese medicines, focusing on the minor components [20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30],we carried out on detailed chemical analysis of an aqueous extract of the I. indigotica roots and have reported characterization of 28 new alkaloids, including a pair of indole alkaloid enantiomers containing dihydrothiopyran and 1,2,4-thiadiazole rings,a pair of bisindole alkaloid enantiomers,and 12 glycosidic indole and bisindole alkaloids,as well as 54 known compounds including 33 constituents isolated from I. indigotica for the first time [31, 32, 33, 34, 35, 36]. Biological assays showed that some of these compounds showed antiviral activity against influenza virus A/Hanfang/359/95 (H3N2) or Coxsackie virus B3 and protective activity against dl-galactosamine- induced hepatocyte (WB-F344 cell) damage. Further examination of the same extract led to separation of unusual stereoisomers of 3,5-bis(2-hydroxybut-3-en-1-yl)-1,2,4-thiadiazole (Fig. 1),named insatindigothiadiazoles A-D (1a-1d). Herein, we report details of the isolation,structure elucidation,postulated biogenetic pathway,and biological activity of the stereoisomers.

Fig. 1. Structures of 1a-1d.

2. Experimental 2.1. General experimental procedures

Optical rotations were measured on a P-2000 polarimeter. UV spectrum was recorded on a V-650 spectrometer. CD spectra were measured on a JASCO J-815 CD spectrometer. IR spectrum was recorded on a Nicolet 5700 FT-IR Microscope spectrometer (FT-IR Microscope Transmission). 1D- and 2D-NMR spectra were obtained at 500 or 600 for 1H and 125 or 150 MHz for 13C, respectively,on a Varian 600 MHz spectrometer or a Bruker 500 MHz spectrometer with solvent peaks as references. ESIMS and HR-ESIMS data were obtained on an AccuToFCS JMS-T100CS spectrometer. Column chromatography (CC) was performed with HPD-110 (Cangzhou Bon Absorber Technology Co. Ltd,Cangzhou, China),MCI resin (120 μm,Mitsubishi Chemical Inc.,Tokyo, Japan),silica gel (200-300 mesh,Qingdao Marine Chemical Inc., China),or RP silica gel (Grace Davison Discovery Science,Deerfield, USA). HPLC separation was performed on a system consisting of a Waters 600 controller,a Waters 600 pump,and a Waters 2487 dual λ absorbance detector with an Alltima (250×10 mm) preparative column packed with C18 (5 μm),an analytical chiral column (Chiralpak AD-H,250 mm×4.6 mm),or a semi-preparative chiral column (Chiralpak AD-H,250 mm×10 mm) packed with amylose tris-(3,5-dimethylphenylphenylcarbamate) coated on 5 μm silica-gel. TLC was conducted on the precoated silica gel GF254 plates. Spots were visualized under UV light (254 or 365 nm) or by spraying with 10% H2SO4 in 95% EtOH followed by heating.

2.2. Plant material

The roots of I. indigotica were purchased in Anhui province, China,in December 2009. The plant was identified by Mr. Lin Ma (Institute of Materia Medica,Beijing 100050,China). A voucher specimen (No. ID-S-2385) was deposited at the Herbarium of the Department of Medicinal Plants,Institute of Materia Medica, Beijing,China.

2.3. Extraction and isolation

The air-dried and pulvarized plant material (50 kg) was decocated with H2O (150 L; 3×1 h). The aqueous extracts were combined and evaporated under reduced pressure to yield a dark-brown residue (32 kg). The residue was dissolved in H2O (122 L),loaded on a macroporous adsorbent resin (HPD-110,19 kg) column (20×200 cm),and eluted successively with H2O (50 L), 50% EtOH (125 L),and 95% EtOH (100 L) to yield three corresponding fractions A,B and C. After removing the solvent under reduced pressure,fraction B (0.9 kg) was separated by CC over MCI resin (5 L),with successive elution using H2O (10 L),30% EtOH (30 L), 50% EtOH (20 L),95% EtOH (10 L),and Me2CO (8 L),to give fractions B1-B5. Fraction B2 (547 g) was subjected to CC over silica gel, with elution by a gradient of increasing MeOH concentration (0-100%) in EtOAc and then with 30% EtOH,to yield subfractions B2-1-B2-5 based on TLC analysis. Subfraction B2-1 (16.3 g) was chromatographed over Sephadex LH-20 with elution by a petroleum ether-chloroform-methanol (5:5:1,v/v/v) mixture to yield B2-1-1-B2-1-10,of which B2-1-2 (600 mg) was further fractionated by RP flash CC with a gradient of increasing MeOH concentration (0-100%) in H2O to yield B2-1-2-1-B2-1-2-4. Fraction B2-1-2-1 (125.5 mg) was purified by RP HPLC (63% MeOH in H2O,flow rate 2.0 mL/min) to give 1 (20.1 mg,tR = 15.4 min). Subsequent separation of 1 by HPLC,using a Chiralpak AD-H column (250×10 mm) packed with amylose tris-(3,5-dimethylphenylphenylcarbamate) coated on 5 μm silica-gel and iPrOH-nhexane mixture (20:80,flow rate 2 mL/min) as the mobile phase, yielded 1a (4.1 mg,tR = 37.0 min),1b (8.2 mg,tR = 20.9 min),1c (2.0 mg,tR = 17.4 min),and 1d (4.2 mg,tR = 16.3 min).

3,5-Bis(2-hydroxybut-3-en-1-yl)-1,2,4-thiadiazole (1): Colorless gum;[α]20D-7.7 (c 1.2,MeOH);UV(MeOH)λmax (log ε): 204 (4.38),235 (4.37) nm; IR (cm-1): νmax 3325,2962,2923,1643,1491,1414,1201,1095,1022,923,799; The data of 1H NMR (acetone-d6, 600 MHz;DMSO-d6,500 MHz) and 13C NMR (acetone-d6,150 MHz; DMSO-d6,125 MHz) were detailed in Table S3 in Supporting information; (+)-ESIMS m/z 249 [M+Na]+; (+)-HR-ESIMS m/z 227.0857 [M+H]+ (calcd. for C10H15N2O2S,227.0849).

(+)-(2'S,2"R)-3,5-Bis(2-hydroxybut-3-en-1-yl)-1,2,4-thiadiazole (insatindigothiadiazole A,1a): Colorless gum; [α]20D+21.3 (c 0.50, MeOH); CD (MeOH) 210 (△ε+1.09),234 (△ε+0.67) nm. 1H NMR (acetone-d6,600 MHz) data and 13C NMR (acetone-d6,150 MHz) data,see Table 1; (+)-HR-ESIMS m/z 227.0849 [M+H]+(calcd. for C10H15N2O2S,227.0849),249.0670 [M+Na]+(calcd. for C10H14N2O2SNa,249.0668).

Table 1
NMR spectroscopic data for 1a-1da.

(-)-(2'S,2"S)-3,5-Bis(2-hydroxybut-3-en-1-yl)-1,2,4-thiadiazole (insatindigothiadiazole B,1b): Colorless gum; [α]20D-25.1 (c 1.10, MeOH); CD (MeOH) 213 (△ε-2.69),236 (△ε-1.44) nm. 1H NMR (acetone-d6,600 MHz) data and 13C NMR (acetone-d6,150 MHz) data,see Table 1; (+)-HR-ESIMS m/z 227.0847 [M+H]+(calcd. for C10H15N2O2S,227.0849),249.0668 [M+Na]+(calcd. for C10H14N2O2SNa,249.0668).

(+)-(2'R,2"R)-3,5-Bis(2-hydroxybut-3-en-1-yl)-1,2,4-thiadiazole (insatindigothiadiazole C,1c): Colorless gum; [α]20D+24.3 (c 0.30, MeOH); CD (MeOH) 214 (△ε+2.39),236 (△ε+1.54) nm. 1H NMR (acetone-d6,600 MHz) data and 13C NMR (acetone-d6,150 MHz) data,see Table 1; (+)-HR-ESIMS m/z 227.0849 [M+H]+(calcd. for C10H15N2O2S,227.0849),249.0667 [M+Na]+(calcd. for C10H14N2O2SNa,249.0668).

(-)-(2'R,2"S)-3,5-Bis(2-hydroxybut-3-en-1-yl)-1,2,4-thiadiazole (insatindigothiadiazole D,1d): Colorless gum; [α]20D-21.1 (c 0.60,MeOH); CD (MeOH) 211 (△ε-1.62),238 (△ε-0.97) nm. 1H NMR (acetone-d6,600 MHz) data and 13C NMR (acetone-d6, 150 MHz) data,see Table 1; (+)-HR-ESIMS m/z 227.0851 [M+H]+ (calcd. for C10H15N2O2S,227.0849),249.0672 [M+Na]+(calcd. for C10H14N2O2SNa,249.0668).

2.4. Synthesis of MPA esters of 1a-1d

To a solution of 1a (1.5 mg),1b (4.0 mg),1c (1.0 mg),or 1d (1.5 mg) in freshly distilled methylene chloride (2mL),was added (R)-(-)- or (S)-(+)-α-methoxyphenylacetic acid [(R)- or (S)-MPA, 3.0 mg),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 4.5 mg),and 4-dimethylaminopyridine (DMAP,4.5 mg). The solution was kept at room temperature overnight then evaporated under reduced pressure to yield a residue. The residue was isolated by preparative TLC (mobile phase: petroleum ether-Me2CO,2:1) to yield 1a-bis-(R)-MPA (1.2mg) or 1a-bis-(S)-MPA (1.3 mg) from 1a; 1b-2"-(R)-MPA (1.1 mg) and 1b-bis-(R)-MPA (1.8mg) or 1b-2"-(S)- MPA(1.2mg) and 1b-bis-(S)-MPA(2.0mg) from 1b; 1c-bis-(R)-MPA (0.8 mg) or 1c-bis-(S)-MPA (0.7 mg) from 1c; and 1d-bis-(R)-MPA (1.1 mg)or 1d-bis-(S)-MPA (1.2 mg) from 1d. 1H NMR (acetone-d6,600 MHz) data for these MPA esters,see Tables S4-S6 in Supporting information.

2.5. ECD calculation

For details see Supporting Information. Briefly,conformational analysis was conducted by Monte Carlo searching with the MMFF94 molecular mechanics force field using Spartan 10 software. The lowest-energy conformers having relative energies within 2 kcal/mol were optimized with the Gaussian 09 program and afforded lowest energy conformers. Subsequently,the conformers were re-optimized using DFT at the B3LYP/6-31+G (d,p) level,with the solvent effects considered using the dielectric constant of MeOH (ε = 32.6) via conductor-like polarizable continuum model (CPCM). The energies,oscillator strengths,and rotational strengths of the first electronic excitations were calculated using the time dependent density functional theory (TDDFT) methodology at the B3LYP/6-311++G (2d,2p) level in vacuum. The re-optimized conformers showed relative Gibbs free energies (△G) under 2 kcal/mol were used for ECD spectra simulation. The ECD spectra were simulated by the Gaussian function (σ = 0.30 eV). To obtain the final spectrum,the simulated spectra of the lowest energy conformations were averaged on the basis of the Boltzmann distribution theory and their relative Gibbs free energy (△G). The final spectrum was inverted to give the corresponding theoretical ECD spectrum of its enantiomer.

3. Results and discussion

The stereoisomer mixture 1 was obtained as a colorless gum with [α]-7.7 (c 1.2,MeOH),which is homogeneous as indicated by thin layer chromatography (TLC) and reversed phase high performance liquid chromatography (RP-HPLC). The positive mode ESIMS of 1 exhibited an adduct molecular ion peaks at m/z 249 [M+Na]+. The molecular formula of C10H14N2O2S,with five degrees of unsaturation,was determined by HR-ESIMS. Although the NMR spectra of 1 in acetone-d6 or DMSO-d6 displayed proton and carbon resonances expected from the formula,several resonances appeared as pairs of signals with chemical shift differences around △δH 0.001-0.008 and △δC 0.02-0.06 (Table S3 and Figs. S7-S18,Supporting information). This suggested that 1 was a mixture of stereoisomers that could not been separated by the conventional TLC and RP-HPLC methods; accordingly,HPLC analysis of 1 using analytical chiral column (Chiralpak AD-H) and n-hexan-iPrOH (4:1) mobile phase displayed four peaks with an integration ratio of about 2:4:1:2 (Fig. S19, Supporting information). Subsequent HPLC separation of 1 using a semi-preparative Chiralpak AD-H chiral column yielded corresponding compounds 1a-1d. The ESIMS and NMR spectroscopic features of 1a-1d (Figs. S20-S38,Supporting information) were similar to those of 1 prior to chiral separation. Detailed comparison of the NMR spectroscopic data of 1a-1d in acetone-d6 demonstrated that the data for 1a and 1d were identical,and as were those for 1b and 1c (Table 1),providing further corroboration that 1 was a mixture of two pairs of enantiomers,1a/1d and 1b/1c. The enantiomeric pairs were further confirmed by specific rotation and circular dichroism (CD) data (Experimental Section and Fig. 2). The planar structure of 1a-1d was elucidated by analysis of the 1D and 2D NMR spectroscopic data of 1a as below.

Fig. 2. The measured CD spectra of 1a (red), 1b (blue dash), 1c (purple dot), and 1d (pink dash dot) and the calculated ECD spectra of (2'S, 2"R)-1 (red dash dot), (2'S, 2"S)-1 (blue short dash), (2'R, 2"R)-1 (purple short dot), and (2'R, 2"S)-1 (pink short dash dot).

The 1H NMR spectrum of 1a in acetone-d6 displayed signals attributable to a pair of vinyl units at δH 5.94/5.97 (each 1H, ddd,J = 17.4,10.8,and 5.4 Hz,H-3'/H-3"),5.24/5.32 (each 1H,ddd, J = 17.4,1.8,and 1.2 Hz,H-4'a/H-4"a),and 5.00/5.10 (each 1H,ddd, J = 10.8,1.8,and 1.2 Hz,H-4'b/H-4"b); a pair of oxygen-bearing methines at δH 4.65/4.51 (each 1H,m,H-2'/H-2"); and a pair of exchangeable secondary hydroxy protons at δH 4.18 (1H,d, J = 5.4 Hz,OH-2') and 4.71 (1H,d,J = 4.8 Hz,OH-2"). It also showed resonances assignable to two methylenes at δH 3.37 (1H,dd, J = 15.6 and 4.2 Hz,H-1"a),3.22 (1H,dd,J = 15.6 and 7.8 Hz,H-1"b), 3.07 (1H,dd,J = 15.6 and 6 Hz,H-1'a),and 3.05 (1H,dd,J = 15.6 and 7.8 Hz,H-1'b). The 13C NMR and DEPT spectra of 1a showed 10 carbon resonances corresponding to the above units,in addition to two additional quaternary carbons at δC 173.7 (C-3),and 188.8 (C-5). These spectroscopic data suggested that 1a was a sulfurcontaining alkaloid having two hydroxybutenyl units and an unusual heterocycle [32],which was further elucidated by 2DNMR data analysis. The HSQC spectrum of 1a furnished assignments of the proton-bearing carbon resonances and corresponding proton resonances in the NMR spectra. The 1H-1H COSY spectrum of 1a showed vicinal homonuclear coupling correlations of H2-1'/H-2'/ H-3'/H2-4',OH-2'/H-2,H2-1"/H-2"/H-3"/H2-4" ,and H-2"/OH-2" (Fig. 3),indicating the presence of 2'-hydroxybut-3'-en-1'-yl and 2"-hydroxybut-3"-en-1"-yl units. The HMBC spectrum of 1a exhibited long-range heteronuclear correlations (Fig. 3) from H2-1' to C-2',C-3' ,and C-3 and from H2-1" to C-2" ,C-3" ,and C-5. These correlations demonstrated that the two units were attached to the sp2 hybrid quaternary carbons C-3 and C-5 in 1a, respectively. The chemical shifts of C-3 (δC 173.7) and C-5 (δC 188.8),along with the molecular composition of 1a (C10H14N2O2S), suggested that the two sp2 hybrid quaternary carbons must be connected by the remaining nitrogen and sulfur atoms to construct a 1,2,4-thiadiazole ring [32]. Accordingly,the planar structure of 1a,as well as the stereoisomers 1b-1d,was determined as 3, 5-bis(2-hydroxybut-3-en-1-yl)-1,2,4-thiadiazole.

Fig. 3. Main 1H-1H COSY (thick lines) and HMBC correlations (arrows, from 1H to 13C) of 1a.

The MPA determination rule based on △δRS values of MPA esters was applied to determine the absolute configurations of the four stereoisomers containing two secondary alcohol groups [37]. Esterification of 1a-1d with (R)- and (S)-MPA gave the corresponding bis esters 1a-bis-(R)-MPA-1d-bis-(R)-MPA and 1a-bis-(S)-MPA- 1d-bis-(S)-MPA,respectively. The monoesters 1b-2"-(R)-MPA and 1b-2"-(S)-MPA were also obtained from the esterification of 1b. The 1H NMR data of the esters were unambiguously assigned through 1H-1H COSY experiments (Figs. S39-S58,Supporting information). The △δRS values (δR-δS) for each pair of (R)-MPA and (S)-MPA esters were calculated as shown in Fig. 4. The same △δRS values with reversed signs for the bis esters of 1a and 1d,as well as of 1b and 1c,confirmed the enantiomeric relationship of the two compound pairs. The △δRS values for the monoesters of 1b completely satisfied the prerequisites of the MPA determination rule for the configurational assignment of secondary alcohols [37] and revealed that 1b had a 2"S configuration. Comparison of △δRS values of the bis esters of 1b with those of the monoesters demonstrated that the MPA moieties at C-2' contributed to the C-2' vinyl protons with positive △δRS values and to the C-2' methylene and 2"-hydroxybut-3"-en-1"-yl protons with negative △δRS values. This suggests that the additive contributions of the MPA moieties at C-2' and C-2" in the bis esters of 1b led to the less positive △δRS values for the vinyl protons and the more negative △δRS values for the methylene protons,respectively,though no △δRS values of the monoesters 1b-2'-(R)-MPA and 1b-2'-(S)-MPA were obtained for comparing the additive contributions. In addition,comparison of △δRS values of the bis esters of 1d with those of the monoesters of 1b demonstrated that the MPA moieties at C-2' contributed to the C-2' vinyl protons with negative △δRS values and to the C-2' methylene and 2"-hydroxybut-3"-en-1"-yl protons with positive △δRS values. The additive contributions of the MPA moieties at C-2' and C-2" in the bis esters of 1d led to the more negative △δRS values for the C-2' vinyl protons and the more positive △δRS values for the C-2" vinyl protons,as well as led to △δRS values of both the C-2' and C-2" methylene protons with smaller magnitudes and anomalous signs due to anisotropic effects of the MPA moieties [36, 37]. This, combined with the enantiomeric relationship of 1a/1d and 1b/1c, revealed that each of the MPA moieties at C-2' and C-2" in the bis esters contributed △δRS values with the same sign to the protons on one side of the plane of the corresponding MPA ester,but with the opposite sign to the protons on the other side of the plane. Therefore,the MPA determination rule based on the △δRS values of MPA esters [37, 38, 39] is fully valid for the assignment of the absolute configurations of 1a-1d; via application of the rule the configurations of 1a-1d were assigned as (2'S,2"R),(2'S,2"S),(2'R,2"R), and (2'R,2"S) (Fig. 4),respectively. The absolute configurations of 1a-1d were also supported by comparison of the experimental CD spectra with the ECD predicted from TDDFT calculations [40]. The theoretically calculated ECD spectra of (2'S,2"R)-,(2'S,2"S)-, (2'R,2"R)-,and (2'R,2"S)-1 were in agreement with the experimental CD spectra of 1a-1d (Fig. 2). Thus,the structures of compounds 1a-1d were determined as (+)-(2'S,2"R)-,(-)-(2'S,2"S)-,(+)- (2'R,2"R)-,and (-)-(2'R,2"S)-3,5-bis(2-hydroxybut-3-en-1-yl)- 1,2,4-thiadiazole,respectively,and named insatindigothiadiazoles A-D.

Fig. 4.δRS values (δR-δS, black data in ppm) for 2"-(R)-MPA and 2"-(S)-MPA monoesters of 1b and pairs of bis-(R)-MPA and bis-(S)-MPA bis esters of 1a-1d.

Interestingly,comparison of the specific rotation data of 1a-1d indicates that signs of the specific rotations are strongly related to the C-2" configuration and weakly to the C-2' configuration. The 2'R and 2"R configurations have contributions of small and large positive values to the specific rotations,respectively,whereas the S configurations have corresponding negative contributions. Further comparison of the experimental CD spectra 1a-1d (Fig. 2) shows that signs of the Cotton effects are also predominated by the C-2" configuration. The 2'R and 2"R configurations contribute small and large positive Cotton effects to the CD spectra,respectively. In contract the S configurations contribute corresponding negative Cotton effects. Altogether,the chiral unit at C-5 has a larger contribution than that of the same unit at C-3 to optical properties of the stereoisomers of 3,5-bis(2-hydroxybut-3-en-1-yl)-1,2, 4-thiadiazole.

Compounds 1a-1d are the first example of natural products with the four stereoisomers being simultaneously isolated and stereo-chemically determined [41]. This discovery provides support for the postulated biosynthetic pathways for the stereoisomers of {3"-[2"'-hydroxybut-3"'-en-1"'-yl]-1",2",4"-thiadiazol- 5"-yl}-5',6'-dihydrospiro[indoline-3,2'-thiopyran]-2-one in our previous paper [32]. Consequently,the stereoisomers 1a-1d are postulated to be biosynthesized from the precursors epiprogoitrin (2) and progoitrin (3),which are abundant in a 2:1 ratio of 2:3 in I.indigotica (Scheme 1) [32]. Myrosinase catalyzed hydrolysis of the precursors liberates unstable intermediates 2a and 3a,respectively, which further produce imidothioates (2'a and 3'a) and nitriles (2"a and 3"a) [42]. Coupling condensation of 2'a with 2"a and 3"a,as well as of 3'a with 2"a and 3"a,would generate 1b,1d,1a,and 1c,respectively. As previously mentioned,the epimeric precursors exist in a 2:1 ratio in this plant [39]; the 2:4:1:2 ratio of 1a:1b:1c:1d is thus consistent with the probability of condensation between the available imidothioates and nitriles. This supports not only the postulated biosynthetic pathway but also the configuration assignment of the four stereoisomers.

Scheme. 1. The plausible biosynthetic pathways of 1a-1d.

In the preliminary in vitro assays [31, 32, 33, 34, 35],compounds 1a and 1b showed antiviral activity against Coxsackie virus B3 with IC50 values of 4.3 and 24.1×10-6 mol/L and SI values of 25.4 and 4.2,respectively (the positive control ribavirin gave IC50 = 0.002×10-6 mol/L and SI = 3.9); however,isomers 1c and 1d were inactive (IC50 values > 10-4 mol/L). Compound 1c inhibited the herpes simplex virus 1 (HSV-1) with IC50 and SI values of 33.3×10-6 mol/L and 3.0,respectively (the positive control acyclovir gave IC50 = 0.4×10-6 mol/L and SI = 241.9),whereas 1a,1b,and 1d were inactive (IC50 values > 10-4 mol/L). In addition,compound 1d inhibited the influenza virus A/Hanfang/359/95 (H3N2),with IC50 and SI values of 19.2×10-6 mol/L and 5.2,respectively (the positive control oseltamivir gave IC50 = 1.6×10-6 mol/L and SI = 777.8), but the other three stereoisomers were inactive (IC50 values > 10-4 mol/L). This indicated that the selective antiviral activities of 1a-1d were highly dependent upon the configurations at C-2' and C-2". In addition,the stereoisomers were also assessed for activities against HIV-1,aswell as against several human cancer cell lines,but were inactive at 10-4 mol/L.

4. Conclusion

From an aqueous extract of I. indigotica roots,the whole four stereoisomers of 3,5-bis(2-hydroxybut-3-en-1-yl)-1,2,4-thiadiazole named insatindigothiadiazoles A-D (1a-1d) were isolated and stereo-chemically characterized. This is the first example of natural products with the four stereoisomers being simultaneously isolated and stereo-chemically determined. To the optical properties of these stereoisomers,the chiral unit at C-5 has a larger contribution than that of the same unit at C-3. The configuration assignment of 1a-1d and the postulated biosynthetic pathway were supported by the co-occurrence of the possible precursors epiprogoitrin and progoitrin in a proper ratio. The stereoisomers displayed selective antiviral activities against influenza virus A (H3N2),Coxsackie virus B3,and HSV-1,while selectivity of the antiviral activities was dependent upon the configurations. This result,combined with our previous studies [32, 33, 34, 35, 36],further illustrate that the chemical constituents with diverse structure types contribute toward pharmacological efficacy that supports the conventional applications of ban lan gen. The new structures provide a further framework for synthesis and in-depth biological evaluation. In particular,the plausible biosynthetic pathway associated to the co-occurring precursors provides an important clue for further studies of biomimetic and total synthesis,chemical transformation,structural modification and structure-activity relationships,as well as biosynthesis of the 1,2,4-thiadiazole alkaloids from this medicinal plant.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at

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