Bioactive Dimeric Carbazole Alkaloids from Murraya koenigii

Article pubs.acs.org/jnp

Bioactive Dimeric Carbazole Alkaloids from Murraya koenigii Chokkalingam Uvarani,†,§ Mathan Sankaran,† Nanjundan Jaivel,‡ Kumarasamy Chandraprakash,† Athar Ata,*,§ and Palathurai S. Mohan*,† †

Department of Chemistry, School of Chemical Sciences, Bharathiar University, Coimbatore, 641 046, Tamil Nadu, India Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, 641 003, Tamilnadu, India § Department of Chemistry, Richardson College for the Environmental and Science Complex, The University of Winnipeg, 599 Portage Avenue, Winnipeg, Manitoba, Canada R3B 2G3 ‡

S Supporting Information *

ABSTRACT: Phytochemical studies on the CHCl3 extract of the fruit pulp of Murraya koenigii afforded three new dimeric carbazole alkaloids, bisgerayafolines A−C (1−3). Bisgerayafolines A−C (1−3) are structurally unique dimeric carbazole alkaloids comprising geranyl moieties incorporated in their structures. Compounds 1−3 exhibited various levels of antioxidant, anti-αglucosidase, DNA binding, and cytotoxic activities and protein interactions. alkaloids displaying DNA intercalative antitumor activity.11 The study of the relationships between the DNA binding properties and the biological activity of carbazoles may lead to a better understanding of the mechanisms involved. On the other hand, drug interactions at the protein binding level significantly affect the apparent distribution volume and their elimination rate. The interactions of compounds with serum albumins have received much attention via studying of antitumor pharmacokinetics and structure−activity relationships. Therefore, it is worthwhile to assess the interaction behavior of isolated dimeric carbazole alkaloids with calf thymus DNA (CTDNA) and bovine serum albumin (BSA) protein, to correlate with their cytotoxic studies. With the rising cost of the plant collections and preparation of crude extracts, it has become necessary for natural product chemists to expand screening targets.12 These facts prompted us to evaluate compounds 1−3 for antioxidant, anti-α-glucosidase, DNA binding, and cytotoxic activities and interactions with bovine serum albumin (BSA).

Murraya koenigii L. Spreng (Rutaceae) (L) is one of the most popular spice species, and its aromatic leaves are being used as an integral part of food additives in India, and more importantly secondary metabolites from all parts of the tree are found to be biologically active. The pharmacological action and the therapeutic efficacy of M. koenigii is mainly due to the presence of carbazole alkaloids.1 Many of these alkaloids possess various biological activities including antimalarial, antioxidant, cytotoxic, anti-HIV, antimicrobial, antidiarrheal, and anti-inflammatory activities.2−4 Fruits are edible, and its juice is used as a kidney pain reliever.5 Although many compounds have been reported from various parts of this plant,6,7 a comprehensive characterization of bioactive compounds from the fruit pulp is still untapped. Our recent chemical investigation of the fruit pulp of M. koenigii extracted with CHCl3 resulted in the isolation of three novel dimeric carbazole alkaloids, bisgerayafolines A−C (1−3), containing geranyl moieties incorporated in their structures. Compounds with α-glucosidase inhibitory and free radical scavenging activities are considered to be an efficient way to treat degenerative diseases including type 2 diabetes,8 cancer,9 and viral infections.10 Moreover DNA is a remarkable bioreceptor for a vast number of small molecules, and it remains a major biological target for the design of anticancer agents. DNA-intercalating agents have been studied for several decades, and a few representatives (ellipticine, rebeccamycin, anthracyclines, acridines, and anthraquinones) are routinely used for the treatment of cancer. Among them ellipticine and rebeccamycin are naturally occurring carbazole © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Bisgerayafoline A (1) was obtained as an off-white, amorphous powder. The UV spectrum showed absorptions at 222, 233, 245, and 315 nm, which are typical for an angular pyrano carbazole skeleton.13 The IR spectrum displayed absorption bands at 3443 (OH), 3360 (NH), and 1079 (C−O) cm−1. The Received: July 3, 2012

A

dx.doi.org/10.1021/np300464t | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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8.37 indicated the presence of a dimeric carbazole alkaloid skeleton in compound 1. The methyl group substitutions at C-5 and C-5″ were inferred by the HMBC interactions of H-6 (δ 7.76) and H-6″ (δ 7.61) with C-5/CH3 (δ 17.9) and C-5″/CH3 (δ 16.3), respectively. This was further confirmed by the NOESY spectrum (Figure 1) that exhibited association of H-6 and H-6″ with C-5/CH3 (δ 17.9) and C-5″/CH3 (δ 16.3), respectively. The HMBC correlation between OCH3 (δ 3.80) and C-9 (δ 154.5) suggested the substitution of the OCH3 group at C-9. This was further supported by an NOE association between OCH3 (δ 3.80) and H-8 (δ 6.82). H-10″ (δ 7.75) showed HMBC interactions with C-8″ (δ 153.5), C-9″ (δ 108.9), and C-7″ (δ 110.9), while H-9″ (δ 6.87) exhibited cross-peaks with C-8″ (δ 153.5), C-10 (δ 103.9), and C-6b″ (δ 118.8), respectively. The NOESY and ROESY spectra (Figure 1) featured cross-peaks of C-8″/OH proton (δ 9.12) with H-9″ (δ 6.87). The HMBC spectrum showed long-range couplings of C-8″/OH with C-10 (δ 103.9) and C-9″ (δ 108.9). These observations and the absence of resonances for C-10 and C-7″ protons in the 1H NMR spectra revealed the C-10−C-7″ linkage between the two carbazole moieties, as in bispyrayafoline.14 The electronic circular dichorism (ECD) spectrum of 1 showed a negative Cotton effect at 235 nm and a positive Cotton effect at 218 nm (an “A-type” curve according to Steglich), indicating that 1 has an aR-biphenyl configuration;15 however with these data the absolute configurations at C-3 and C-3″ of the (2H) pyran rings remain undefined.15 On the basis of these spectroscopic studies, the structure of 1, a new dimeric carbazole alkaloid, bisgerayafoline A, was defined as 3,3′bis[(E)-3,7-dimethylocta-2,6-dienyl]-9′-methoxy-3,3′,5,5′-tetramethyl-3,3′,11,11′-tetrahydro-7,10′-bipyrano[3,2-a]carbazol-8ol. Bisgerayafoline B (2) was isolated as off-white flakes. Its UV, IR, and 1H and 13C NMR data were similar to those of compound 1, with the exception of three resonances due to C7″/phenolic, N-11, and N-11″ amine protons. The 1H NMR spectrum of 2 showed one-proton singlets at δ 5.23, 7.66, and 7.50 due to the 7″-OH, 11-NH, and 11″-NH protons, respectively. The 1H NMR spectrum also showed two doublets at δ 6.98 (H-9″) and 7.91 (H-10″), which exhibited HMBC correlations with C-8″ (δ 99.9), C-6b″ (δ 118.8), and C-7″ (δ 151.7). The absence of protons for C-10 and C-8″ in the 1H NMR spectrum and the aforementioned NMR observations revealed the linkage of two carbazole moieties via a C-8″−C-10 single bond. This linkage was supported by the NOESY correlation of the 7″-OH proton (δ 5.23) with the 11-NH proton (δ 7.66). The COSY and NOESY interactions of compound 2 are shown in Figure 1. The complete 1H and 13C NMR chemical shift assignments and 1H/13C one-bond shift correlations of 2, as determined from the HSQC spectrum and HMBC correlations, are presented in Table 1. The ECD spectrum indicated that 2 also had an aR-biphenyl configuration.15 These spectral data led us to assign the structure of 2 to a new dimeric carbazole alkaloid, bisgerayafoline B, which was characterized as 3,3′-bis[(E)-3,7-dimethylocta-2,6-dienyl]9′-methoxy-3,3′,5,5′-tetramethyl-3,3′,11,11′-tetrahydro-8,10′bipyrano[3,2-a]carbazol-7-ol. Bisgerayafoline C (3) was purified as an off-white powder. The UV spectrum was similar to those of compounds 1 and 2. The IR spectrum of 3 did not exhibit a broad absorption band at 3350−3460 cm−1, suggesting the absence of a hydroxy group. The HRFABMS showed the molecular ion at m/z 843.5098 [M]+, which was consistent with the molecular formula

HRFABMS showed a pseudomolecular ion peak [M + H]+ at m/z 815.4780, and NMR data established its molecular formula as C55H62N2O4. The 13C NMR spectrum (DMSO-d6, 100 MHz) (Table 1) showed resonances of 46 carbons. A combination of 13C APT and HSQC data suggested overlapped resonances for nine carbons. The data further revealed the presence of 55 carbons comprising 11 methyl, 14 methine, six methylene, and 24 quaternary carbons. Five of the oxygenated quaternary carbons resonated at δ 154.5, 153.5, 148.1, 151.8, and 77.6 and were attributed to C-9, C-8″, C-4a″, C-4a, and C3/C-3″, respectively. The 1H NMR spectrum (DMSO-d6, 400 MHz) (Table 1) indicated the presence of two geranyl units, as evidenced by the resonances at δ 5.02 (1H, t, J = 5.4, 9.4 Hz), 5.08 (1H, t, J = 5.4, 9.4 Hz), 1.70−1.75 (6H, m), 3.40 (2H, m), 4.84 (1H, t, J = 6.0, 8.7 Hz), 2.1 (2H, m), and four methyl singlets. A set of AB-type doublets, integrating for two protons each at δ 7.06 (J = 12.8 Hz) and 5.50 (J = 12.8 Hz), was indicative of the presence of two (2H)-pyran rings. The HMBC spectrum exhibited interactions of the C-2 olefinic proton (δ 5.50) with C-1′/C-1‴ (δ 23.8), C-11b (δ 105.3), and C-3 (δ 77.6), whereas the 3a/3a″ methyl group (δ 1.35) showed HMBC cross-peaks with C-3 (δ 77.6) and C-2 (δ 127.5) (Table 1). These HMBC data suggested attachment of the two geranyl moieties at C-3 and C-3″ of the (2H)-pyran rings. The signals for two aryl methyls [δ 2.40 (C-5/CH3), 2.30 (C-5″/ CH3)], two pairs of ortho-coupled protons [δ 7.80 (d, J = 8.7 Hz, H-7), 7.75 (d, J = 8.0 Hz, H-10″), 6.87 (d, J = 8.0 Hz, H9″), and 6.82 (d, J = 8.7 Hz, H-8)], two singlets at δ 7.76 (H-6) and 7.61 (H-6″), methoxy protons at δ 3.80 (C-9/OCH3), a phenolic proton at δ 9.12, and two amine protons at δ 9.93 and B



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Table 1. 1H and 13C NMR Assignments and HMBC Correlations for Compounds 1, 2, and 3 1 positions

δC(APT)

δH (J = Hz)

2 HMBC

δC(APT)

1 (1″)

119.9, CH

7.06 d (12.8)

3, 11b, 11a

117.3, CH

2 (2″)

127.5, CH

5.50 d (12.8)

11b, 3, 1′

128.6, CH

3 (3″) 4a 5 6 6a 6b 7 8 9 10 10a 11a 11b (11b″) 4a″ 5″ 6″

77.6, 151.8, 116.6, 117.1, 117.5, 117.7, 119.3, 104.5, 154.5, 103.9, 141.1, 135.5, 105.3, 148.1, 116.3, 120.0,

C C C CH C C CH CH C C C C C C C CH

6a″ 6b″ 7″ 8″ 9″ 10″ 10a″ 11a″ 1’ (1‴) 2’ (2‴)

116.1, 118.8, 110.9, 153.5, 108.9, 120.2, 140.2, 139.7, 23.8, 122.5,

C C C C CH CH C C CH2 CH

3′ (3‴) 4′ 5′ 6′ 7′ 4‴ 5‴ 6‴ 7‴ 3,3″-CH3 5-CH3 9-OCH3 3′,3‴-CH3 7′-CH3 7‴-CH3 7′-CH3 7‴-CH3 5″-CH3 8″-OH 11-NH

135.1, 40.7, 22.7, 124.5, 131.1, 39.3, 26.4, 124.6, 130.7, 25.8, 17.9, 56.7, 15.7, 17.8, 17.8, 25.8, 25.8, 16.3,

C CH2 CH2 CH C CH2 CH2 CH C CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

11″NH,CH3 7″-OH 9″-OCH3

7.76 s

5a, 4a

7.80 d (8.7) 6.82 d (8.7)

9, 10a, 10 7″, 9, 7

7.61 s

6b″, 5a″, 4a″

6.87 d (8.0) 7.75 d (8.0)

8″, 10, 6b″ 7″, 8″, 9″

3.40 m 5.02 t (6.4, 9.4)

2′, 3′ 4‴, 1′, 3a′

1.70−1.75 m 2.1 m 4.84 t (6.0, 8.7)

7′, 6′, 3a′ 3a′ 4′, 7a′

1.70−1.75 m 1.70−1.75 m 5.08 t (5.4, 9.4)

6‴, 3a‴ 6‴, 7b‴ 7a‴, 5‴

1.35 2.40 3.80 1.38 1.51 1.41 1.60 1.51 2.30 9.12 9.93

3, 2 4a, 6, 5 9 3′, 2′, 3‴ 7b′, 7′, 6′ 7b‴, 6‴ 7a′, 7′, 6′ 7b‴, 6‴ 4a″, 6″ 8″, 9″, 10 10a, 6b, 11a 6b″, 6a″

s s s s s s s s s s s

8.37 s

78.0, 150.2, 99.3, 122.4, 117.6, 118.3, 117.0, 105.0, 154.9, 111.2, 138.0, 140.5, 104.5, 149.3, 99.3, 120.4,


117.3, 118.8, 151.7, 99.9, 108.9, 121.2, 139.0, 134.7, 23.8, 121.6,

C C C C CH CH C C CH2 CH

136.8, C 40.8, CH2 22.8, CH2 124.2, CH 131.9, C 39.1, CH2 26.3, CH2 124.1, CH 131.6, C 25.6, CH3 16.1, CH3 57.0, CH3 15.4, CH3 17.57, CH3 17.52, CH3 25.6, CH3 25.6, CH3 16.7, CH3

δH (J = Hz) 6.39 dd (4.0, 10.0) 5.5 dd (4.0, 10.0)

3 δC(APT)

HMBC 11b, 2, 4a″

118.9, CH

11b, 1, 3, 1′

127.8, CH

7.83 s

6b, 5, 11a, 4a

7.78 d (8.5) 6.83 d (8.5)

10, 10a, 9 10, 6b, 9

7.60 s

5″, 11a″, 4a″

6.98 d (8.0) 7.91 d (8.0)

8″, 6b″, 7″ 10a″, 8″, 7″

3.42 m 5.18 t (6.5, 7.0)

2′, 3‴, 3′, 2‴ 1′, 1‴, 4′, 3a‴

1.69 m 2.11 m 5.18 t (6.5, 7.0)

6′, 7b′ 6′, 7′, 4′ 5′, 7a′

1.51 m 1.64 m 4.75 t (6.5, 7.0)

6‴, 7‴ 6‴, 7‴, 3‴ 7a‴, 7b‴

1.38 2.48 3.88 1.28 1.55 1.42 1.65 1.55 2.32

2, 3 5, 6a, 6, 4a C-9 2′, 3′, 4‴ 7b′, 7′ 7‴, 7b‴, 6‴ 7a′, 7′ 7a‴, 7‴ 4a″, 6″, 11a″

s s s s s s s s s

7.66 s

6a, 11a

7.50 s

6a″, 11a″, 10a″ 8″, 6a″, 10″

5.23 s

78.0, 150.0, 117.0, 121.0, 119.1, 118.3, 117.1, 104.5, 155.2, 110.5, 140.5, 139.6, 106.0, 154.9, 119.5, 120.0,


117.5, 115.5, 122.2, 115.4, 156.3, 91.8, 142.1, 136.0, 23.5, 122.1,

C C CH C C CH C C CH2 CH

136.2, 39.5, 22.8, 124.2, 130.9, 40.2, 26.5, 124.2, 131.2, 25.5, 16.2, 56.5, 15.8, 17.5, 17.5, 25.8, 25.8, 17.2,

C CH2 CH2 CH C CH2 CH2 CH C CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

δH (J = Hz)

HMBC

7.23 dd (10.0, 1.5) 5.70 dd (10.0, 1.5)

11b, 3, 4a

7.59 s

5, 4a

7.80 d (8.5) 6.85 d (9.0)

10a, 9 10, 7

7.78 s

6a″, 11a″, 4a″

7.93 s

9″, 10a″, 6a″

6.98 s

8″, 10a″, 9″

3, 11b

3.52 m 5.19 t (7.0, 7.5)

1.73 m 2.20 m 5.15 m

3a′, 3′ 3a′

1.83 m 1.73 m 4.86 m

5‴ 7a‴, 3‴

1.56 2.32 3.91 1.53 1.62 1.45 1.69 1.69 2.51

s s s s s s s s s

6b, 6, 4a 9 4‴, 6‴ 7b′, 6′, 7′ 7b‴, 6‴, 7‴ C-7a′, C-6’ 7a‴, 6‴ 4a″, 5a″, 6″

7.69 s

6b, 10a

33.5, CH3

4.09 s

11a″, 10a″, 11c″

55.5, CH3

3.86 s

9″

spectrum showed two three-proton singlets at δ 3.86 and 4.09 as well as two one-proton singlets at δ 7.93 (H-7″) and 6.98 (H-10″). Signals at δ 3.86 and 4.09 showed HSQC cross-peaks

C57H66N2O4. The 1H NMR spectrum (Table 1) of 3 was also akin to that of compound 2, showing the lack of signals at δ 7.66 (11″-NH) and 5.23 (7″-OH). Instead, the 1H NMR C



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Figure 1. Selected 2D correlations of compounds 1 and 2.

Table 2. Anti-oxidant and Anti-α-glucosidase Data of Compounds (1−3)a IC50 (μM)e sample 1 2 3 rutin BHT acarbose

FRAPb 5886.7 5428.9 3798.9 7794.4 8345.6

± ± ± ± ±

147.7 270.5 16.8 22.1 87.8

metal chelatingc 7.3 9.2 6.5 18.5 15.4

± ± ± ± ±

0.6 0.5 0.1 0.6 0.6

DPPH•

ABTS+•d 4319.9 6169.5 3661.8 10106.7 6052.7

± ± ± ± ±

5.8 6.8 5.8 2.3 3.2

31.9 31.1 41.8 4.7 4.5

± ± ± ± ±

3.3 2.9 1.9 0.3 0.2

OH• 12.3 8.8 13.2 19.4 11.5

± ± ± ± ±

4.0 0.4 1.6 5.3 3.1

NO•

α-glucosidase

± ± ± ± ±

45.4 ± 1.8 41.2 ± 1.6 69.0 ± 1.4

6.5 7.2 8.6 4.7 8.6

2.4 1.6 1.0 0.7 4.1

15.2 ± 0.6

“±” represents standard error of these bioassays. bmmol Fe(II)/g. cmg EDTA equivalent/g. dμmol trolox equivalent/g. eIC50 value represents the concentration of compounds required to inhibit 50% of the activity. Rutin and BHT were used as positive controls in the antioxidant assay, while acarbose was used as a positive control in the α-glucosidase inhibition assay. a

with carbons resonating at δ 55.5 (C-9″/OCH3) and 33.5 (N11″/CH3), respectively. The N-methyl protons (δ 4.09) showed HMBC cross-peaks with the signals at C-10a″ (δ 142.1) and C-11a″ (δ 136.0). These HMBC spectral data suggested the substitution of a methyl group at N-11″. H-7″ (δ 7.93) and H-10″ (δ 6.98) exhibited HMBC correlations with C8″ (δ 115.4), C-9″ (δ 156.3), and C-10a″ (δ 142.1). These NMR spectroscopic data and the absence of C-10 and C-8″ protons in the 1H NMR spectrum indicated the linkage of two carbazole moieties through a C-8″−C-10 single bond as reported in bismahanine.14 Compound 3 showed a mirror image Cotton effect couplet (positive at 239 nm and negative at 221 nm) compared to those of 1 and 2, indicating that 3 has an aS-biphenyl configuration.15 On the basis of these spectroscopic studies, the structure of 3, a new dimeric carbazole alkaloid, bisgerayafoline C, was defined as 3,3′-bis[(E)-3,7-

dimethylocta-2,6-dienyl]-9,9′-dimethoxy-3,3′,5,5′,11-pentamethyl-3,3′,11,11′-tetrahydro-8,10′-bipyrano[3,2-a]carbazole. A few dimeric carbazole alkaloids have been reported.2b,3b,7a,d,16 Compounds 1−3 represent a new series of dimeric carbazole alkaloids containing geranyl moieties incorporated in their structures. Compounds 1−3 were evaluated for their in vitro antioxidant (FRAP, metal chelating, ABTS+•, DPPH•, OH•, and NO•) and α-glucosidase inhibitory activities17 (Table 2). All the compounds were moderately active in the aforementioned bioassays. The DNA binding properties were evaluated by absorption, fluorescence spectrometric titration, and competitive ethidium bromide (EB) displacement assays. The UV−visible titration curve of compounds 1−3, upon increasing concentrations of CT-DNA, exhibited a red shift with a profound hypochromic effect (54−82%) (Figures 2 and S1). The extent of D



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Figure 2. (a) Changes in electronic absorption spectra of compound 1 (MK1) (50 μM) with increasing concentrations (0−25 μM) of CT-DNA. (b) Fluorescence quenching curves of ethidium bromide bound to CT-DNA (5 μM) with increasing concentration of compound 1 (MK1) (0−50 μM). (c) Emission spectra of BSA (2 μM) in the presence of increasing amounts of compound 1 (MK1) (0−2.5 μM). (d) Synchronous spectra of BSA (2 μM) in the presence of increasing amounts of compound 1 (MK1) (0−2.5 μM) at a wavelength difference of Δλ = 60 nm.

The resulting decreases in fluorescence intensity (Figures 2 and S3 in the SI) of the DNA−EB system with increasing concentration of compounds indicated that the EB molecules were displaced by 1−3 from their DNA binding sites. The quenching data were analyzed using the Stern−Volmer equation;21 the calculated quenching constant (Kq) and the apparent binding constant (Kapp) values (Table 4) suggested

hypochromism is commonly consistent with the strength of the intercalative binding interaction. In order to compare quantitatively the binding strength of the compounds, the intrinsic binding constant (Kb) was calculated. The calculated intrinsic binding constants from absorption titration18 (Table 3) and the increasing emission intensity of compounds 1−3 Table 3. Binding Constant (Kb) and Photometric Properties of Compounds 1−3 in Contact with CT-DNA compound

λmax (nm)

change in shift (nm)

hypochromicity Δε (%)

Kb × 10 (M−1)

1 2 3

259 305 306

red shift (1) red shift (2) red shift (1)

82 55 54

4.3 ± 0.47 2.2 ± 0.11 2.0 ± 0.13

Table 4. Quenching Constant (Kq) and Apparent Binding Constant (Kapp) of Ethidium Bromide Bound to CT-DNA

5

compound


Kq × 105 (M−1)

Kapp × 105 (M−1)

1 2 3

44 22 28

0.32 ± 0.30 0.10 ± 0.09 0.30 ± 0.16

1.60 ± 0.30 0.50 ± 0.09 0.13 ± 0.16

stronger interaction of compounds with CT-DNA,22 which is consistent with the above absorption and emission observations. Compound 1 is more strongly bound to CT-DNA than the other isolates, indicating that the planar carbazole chromophore can intercalate into DNA, while the geranyl residue contacts externally in the minor groove of DNA.23

with the incremental addition of CT-DNA (Figure S2 in the Supporting Information (SI)) in fluorescence studies revealed that they bind to DNA via an intercalative mode.19,20 Competitive binding experiments of compounds 1−3 as quenchers with DNA-bound EB were also carried out to confirm the binding of compounds with DNA via intercalation. E



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Table 5. Quenching Constant (Kq), Binding Constant (Kbin), Number of Binding Sites, and Photometric Properties of Compounds 1−3 in Contact with BSA Protein compound

Kq × 105 (M−1)

Kbin × 104 (M−1)


change in shift (nm)

n

1 2 3

3.52 ± 0.33 2.20 ± 0.21 2.21 ± 0.21

1.02 ± 0.24 0.72 ± 0.19 0.09 ± 0.19

47 36 36

red (4) red (3) blue (1)

0.87 0.67 0.69

Coimbatore and identified by Dr. G. V. S. Murthy from the Botanical Survey of India, Southern Circle, Tamil Nadu, Southern India. A voucher specimen (4408) was deposited in the Department of Botany, Bharathiar University Herbarium, Coimbatore, India. Extraction and Isolation. The shade-dried fruit pulp (1.5 kg) was powdered and extracted with CHCl3 in a Soxhlet apparatus for 12 h. The filtrate was evaporated under reduced pressure to give a residue (200 g), which was loaded onto a silica gel column (60−120 mesh), and the column was eluted with a gradient of a n-hexane−EtOAc (0− 100%), which afforded several fractions, which were pooled based on their analytical TLC results. The fractions obtained with the mixture of n-hexane−EtOAc (92:8 v/v) were further chromatographed with a gradient elution using a mixture of n-hexane−CHCl3 (88:12 and 86:14 v/v) to afford two fractions, labeled as MKF1 and MKF2. Fraction MKF1 was subjected to preparative TLC (PTLC) using n-hexane− CHCl3 (85:15 v/v) as the mobile phase in order to purify compound 2 (6 mg, Rf = 0.76, 0.003%). PTLC of MKF2 using n-hexane−CHCl3 (80:20 v/v) as mobile phase yielded compounds 1 (15 mg, Rf = 0.32, 0.0075%) and 3 (8 mg, Rf = 0.54, 0.004%). Bisgerayafoline A (1): off-white, amorphous powder; [α]20D +1.2 (c 0.25, CHCl3); UV (EtOH) λmax (log ε) 222 (5.65), 233 (5.67), 245 (5.69), 315 (5.79) nm; ECD (EtOH) λ (Δε) 210 (3.5), 218 (15.0), 235 (−12.6); IR (KBr) νmax 3443 (OH), 3360 (NH) 1079(C−O) cm−1; 1H NMR (DMSO-d6, 400 MHz), see Table 1; 13C NMR (DMSO-d6, 100 MHz), see Table 1; HRFABMS m/z 815.4780 [M + H]+ (calcd for C55H63N2O4, 815.4788). Bisgerayafoline B (2): off-white flakes; [α]20D +12.3 (c 0.22, CHCl3); UV (EtOH) λmax (log ε) 220 (5.74), 230 (5.77), 243 (5.70), 317 (5.86) nm; ECD (EtOH) λ (Δε) 212 (1.5), 220 (2.3), 232 (−4.7); IR (KBr) νmax 3350 (OH), 3211 (NH) 1073 (C−O) cm−1; 1H NMR (CDCl3, 500 MHz), see Table 1; 13C NMR (CDCl3, 125 MHz), see Table 1; HRFABMS m/z 815.4775 [M + H]+ (calcd for C55H63N2O4, 815.4788). Bisgerayafoline C (3): off-white powder; [α]20D −12.0 (c 0.20, CHCl3); UV (EtOH) λmax (log ε) 219 (5.79), 232 (5.88), 245 (5.67), 317 (5.88) nm; ECD (EtOH) λ (Δε) 221 (−9.2), 239 (11.8); IR (KBr) νmax 3217 (NH), 1073 (C−O), 1484, 1209, 1025 cm−1; 1H NMR (CDCl3, 500 MHz), see Table 1; 13C NMR (CDCl3, 125 MHz), see Table 1; HRFABMS m/z 843.5098 [M]+ (calcd for C57H66N2O4, 843.5101). Biological Screening of Compounds 1−3. Isolates 1−3 were screened for their potential as antioxidant, anti-α-glucosidase, DNA binding, and cytotoxic activities and interactions with bovine serum albumin. Detailed bioassay procedures have been provided in the Supporting Information.

Qualitative analysis of the binding of chemical compounds to BSA protein is usually detected by fluorescence spectra. The addition of compounds 1−3 to a solution of BSA resulted in a significant decrease of the fluorescence intensity (Figures 2 and S4) accompanied with a blue shift. To study the quenching process further, fluorescence quenching data were analyzed with the Stern−Volmer and Scatchard equations.24 From the Stern−Volmer and Scatchard plots (Figure S5) the calculated quenching constant (Kq), binding constant (Kbin),25 and number of binding sites (n) are given in Table 5. The calculated value of n is around 1 for all of the compounds, indicating the existence of just a single binding site in BSA. The values of Kq and Kbin for the compounds suggested that they have stronger interactions with BSA.26 A simple method to explore the type of quenching is UV−visible spectra of BSA in the absence and presence of the compounds (Figure S6). This showed that the absorption intensity of BSA was enhanced by the addition of compounds, and there was a small blue shift observed. It revealed that there exists a static interaction between BSA and the added compounds.27 The synchronous fluorescence spectra of BSA with various concentrations of test compounds resulted in a small decrease in fluorescence intensity at Δλ = 15 nm and considerable decrease in fluorescence intensity at Δλ = 60 nm (Figures S7 and S8), suggesting that the interaction of compounds with BSA affects the conformation of the tryptophan significantly than tyrosine microregions. Thus, the strong interaction between the compounds and BSA suggested that these compounds can be readily stored in protein and will be released at appropriate targets. Compound 1 exhibited moderate cytotoxicity in the MTT assay against all tested cell lines, namely, human cervical cancer cells (HeLa), stomach adenocarcinoma cells (AGS), and colorectal adenocarcinoma cells (HCT116), with IC50 values of 17.2 ± 0.11, 23.2 ± 0.13, and 22.3 ± 0.25 μM, respectively. Cisplatin, a standard anticancer drug, was used as a positive control and showed IC50 values of 1.32 ± 0.25, 1.67 ± 0.14, and 1.98 ± 0.22 μM, respectively. In summary, we have identified three new dimeric geranylcontaining pyranocarbazole alkaloids. These compounds are moderately active in several bioassays.

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EXPERIMENTAL SECTION

ASSOCIATED CONTENT

S Supporting Information *

General Experimental Procedures. Optical rotations were measured on a Rudolph Autopol IV polarimeter at a wavelength of 50 nm. ECD spectra were recorded at 25 °C on a Jasco-715 spectrophotometer. IR spectra were recorded on a Perkin-Elmer 1600 FT IR spectrophotometer as KBr pellets, and the absorption frequencies are expressed in reciprocal centimeters (cm−1). Electronic absorption spectra were recorded using a Jasco V-630 spectrophotometer. Emission spectra were measured on a Jasco FP 6600 spectrofluorometer. Mass spectral studies were carried out on a Hewlett-Packard 5989B MS. 1D and 2D NMR experiments were performed on a Bruker Avance III spectrometer at 400 MHz. Plant Material. The blue-violet-colored Murraya koenigii ripened fruits were collected during June 2010 from nearby areas of

DNA and protein binding studies and bioassay procedures, as well as copies of the 1H, 13C, and 2D-NMR spectra of compounds 1−3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(A. Ata) Tel: 204-786-9389. Fax: 204-774-2401. E-mail: a. [email protected]. (P. S. Mohan) Tel: +91-422-242-8314. Fax: +91-422-242-2387. E-mail: [email protected]. F



Article

Notes

(9) (a) Berrino, F.; Verdecchia, A.; Lutz, J. M.; Lombardo, C.; Micheli, A.; Capocaccia, R. Eur. J. Cancer 2009, 45, 901−908. (b) Goss, P. E.; Baker, M. A.; Carver, J. P.; Dennis, J. W. Clin. Cancer Res. 1995, 1, 935. (10) (a) Pace, G. W.; Leaf, C. D. Free Radical Biol. Med. 1995, 4, 523−528. (b) Bendich, A. Adv. Exp. Med. Biol. 1990, 262, 1−12. (c) Broek, V.; Leon, A. G. M. Carbohydrate Drug Design; New York, 1997; p 471. (11) (a) Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215−2235. (b) Demeunynck, M.; Bailly, C.; Wilson, W. D. From small molecules to drugs. DNA and RNA Binders; Wiley-VCH: Weinheim, 2002; pp 482−497. (c) Berrino, F.; Verdecchia, A.; Lutz, J. M.; Lombardo, C.; Micheli, A.; Capocaccia, R. Eur. J. Cancer 2009, 45, 901−908. (d) Bush, J. A.; Long, B. H.; Catino, J. J.; Bradner, W. T.; Tomita, K. J. Antibiotics 1987, 40, 668. (e) Rodrigues Pereira, E.; Belin, L.; Sancelme, M.; Prudhomme, M.; Ollier, M.; Rapp, M.; Severe, D.; Riou, J. F.; Fabbro, D.; Meyer, T. J. Med. Chem. 1996, 39, 4471−4477. (12) (a) Mollataghi, A.; Coudiere, E.; Hadi, A. H. A.; Mukhtar, M. R.; Awang, K.; Litaudon, M.; Ata, A. Fitoterapia 2012, 83, 298−302. (b) Kingston, D. G. I. J. Nat. Prod. 2011, 74, 496−511. (13) (a) Chakraborty, D. P. Progress in the Chemistry of Organic Natural Products; Herz, W.; Grisebach, H.; Kirby, G. W., Eds.; Springer-Verlag: New York, 1977; Vol. 34, p 299. (b) Bhattacharyya, D. P.; Chakraborty, D. P. Progress in the Chemistry of Organic Natural Products; Herz, W.; Grisebach, H.; Kirby, G. W., Eds.; Springer-Verlag: New York, 1987; Vol. 52, p 159. (c) Chakraborty, D. P.; Roy, S. Progress in the Chemistry of Organic Natural Products; Herz, W.; Grisebach, H.; Kirby, G. W., Eds.; Springer-Verlag: New York, 1991; Vol. 57, p 71. (14) (a) Yukari, T.; Hiroe, K.; Nordin, H. L.; Nobuji, N. J. Agric. Food Chem. 2003, 51, 6461−6467. (b) Cuong, N. M.; Hung, T. Q.; Sung, T. V.; Taylor, W. C. Chem. Pharm. Bull. 2004, 52, 1175−1178. (15) (a) Botha, J. J.; Ferreira, D.; Roux, D. G. J. Chem. Soc., Perkin Trans. 1 1981, 1235−1245. (b) Shen, Y. C.; Cheng, Y. B.; Lan, T. W.; Liaw, C. C.; Liou, S. S.; Kuo, Y. H.; Khalil, A. T. J. Nat. Prod. 2007, 70, 1139−1145. (c) Chen, M.; Kilgore, N.; Lee, K. H.; Chen, D. F. J. Nat. Prod. 2006, 69, 1697−1701. (d) Ferreira, D.; Marais, J. P. J.; Slade, D. Phytochemistry 2005, 66, 2216−2237. (e) Gill, M.; Steglich, W. Progress in the Chemistry of Organic Natural Products; Springer Verlag: Wien, 1987; Vol 51, pp 1−317. (16) (a) Ito, C.; Wu, T. S.; Furukawa, H. Chem. Pharm. Bull. 1990, 38 (5), 1143−1145. (b) Ito, C.; Furukawa, H. Chem. Pharm. Bull. 1990, 38 (6), 1548−1550. (c) Furukawa, H.; Wu, T. S.; Kuoh, C. S. Chem. Pharm. Bull. 1985, 38 (6), 2611−2613. (d) Ito, C.; Furukawa, H. Chem. Pharm. Bull. 1991, 39 (5), 1355−1357. (e) Wang, Y. S.; He, H. P.; Shen, M. Y.; Hong, X.; Zhao, Q.; Hao, X. J. Chin. Chem. Lett. 2002, 13, 849−850. (f) Furukawa, H.; Wu, T. S.; Ohta, T. Chem. Pharm. Bull. 1983, 31 (11), 4202−4205. (g) Chakraborty, M.; Mukhopadhyay, S. Tetrahedron Lett. 2010, 51, 3732. (h) Wu, T.-S.; Huang, S.-C.; Wu, P.L. Tetrahedron Lett. 1996, 37, 7819. (i) Wu, T.-S.; Wang, M.-L.; Lai, J.S.; Chihiro, I.; Furukawa, H. Phytochemistry 1991, 30, 1052. (17) Methods for antioxidant and anti-α-glucosidase assays are described in the Supporting Information. (18) The intrinsic binding constants (Kb) of compounds with CTDNA were determined from the following equation: [DNA]/(εa − εf) = [DNA]/(εa − εf) + 1/Kb (εb − εf), where [DNA] is the concentration of DNA in base pairs, and the apparent absorption coefficient εa, εf, and εb correspond to Aobs/[compound], the extinction coefficient of the free compound, and the extinction coefficient of the compound when fully bound to DNA, respectively. The plot of [DNA]/(εa − εf) versus [DNA] gave a slope and intercept that are equal to 1/(εb − εf) and 1/Kb(εb − εf), respectively; Kb is the ratio of the slope to the intercept. (19) Tysoe, S. A.; Morgan, R. J.; Baker, A. D.; Strekas, T. C. J. Phys. Chem. 1993, 97, 1707−1711. (20) (a) Haworth, I. S.; Elcock, A. H.; Freemann, J.; Rodger, A.; Richards, W. G. J. J. Biomol. Struct. Dyn. 1991, 9, 23−44. (b) Raja, D. S.; Bhuvanesh, N. S. P.; Natarajan, K. Eur. J. Med. Chem. 2011, 46, 4584−4594.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi, India (05/ 472(0151/2010-EMR-I). Research work at A.A.’s lab was funded by the University of Winnipeg, a grant from the Canadian Commonwealth Scholarship Program, Manitoba Health Research Council, and Manitoba Hydro.

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REFERENCES

(1) Chevallier, A. The Encyclopedia of Medicinal Plants; Dorling Kindersley: London, 1996. (2) (a) Bringmann, G.; Ledermann, A.; Francois, G. Heterocycles 1995, 40, 293. (b) Rahman, A.; Zaidi, R.; Firdous, S. Fitoterapia 1988, LIX, 494−495. (c) Ramsewak, R. S.; Nair, M. G.; Strasburg, G. M.; DeWitt, D. L.; Nitiss, J. L. J. Agric. Food Chem. 1999, 47, 444−447. (d) Chowdhury, B. K.; Jha, S.; Bhattacharyya, P.; Mukherjee, J. Indian J. Chem. 2001, 40B, 490−494. (e) Rahman, M. M.; Gray, A. I. Phytochemistry 2005, 66, 1601−1606. (f) Wang, Y. S.; He, H. P.; Shen, M. Y.; Hong, X.; Hao, X. J. J. Nat. Prod. 2003, 66, 416−418. (g) Meragelman, K. M.; McKee, C. T.; Boyd, R. M. J. Nat. Prod. 2000, 63, 427−428. (3) (a) Fiebig, M.; Pezzuto, J. M.; Soejarto, D. D.; Kinghorn, A. D. Phytochemistry 1985, 24, 3041−3043. (b) Ito, C.; Itoigawa, M.; Nakao, K.; Murata, T.; Tsuboi, M.; Kaneda, N.; Furukawa, H. Phytomedicine 2006, 13, 359−365. (c) Chakraborty, D. P. Fortschr. Chem. Org. Naturst. 1977, 34, 299. (d) Chakraborty, M.; Nath, A. C.; Khasnobis, S.; Chakraborty, M.; Konda, Y.; Harigaya, Y.; Komiyama, K. Phytochemistry 1997, 46, 751−755. (e) Itoigawa, M.; Kashiwada, Y.; Ito, C.; Furukawa, H.; Tachibana, Y.; Bastow, K. F.; Lee, K. H. J. Nat. Prod. 2000, 63, 893−897. (f) Ito, C.; Itoigawa, M.; Aizawa, K.; Yoshida, K.; Ruangrungsi, N.; Furukawa, H. J. Nat. Prod. 2009, 72, 1202−1204. (4) (a) Potterat, O. Curr. Org. Chem. 1997, 1, 415−440. (b) Kato, S.; Kawai, H.; Kawasaki, T.; Toda, Y.; Urata, T.; Hayakawa, Y. J. Antibiot. 1989, XLII, 1879−1881. (c) Iwatsuki, M.; Niki, E.; Kato, S. BioFactors 1993, 4, 123−128. (d) Kato, S.; Shindo, K.; Kataoka, Y.; Yamagishi, Y.; Mochizuki, J. J. Antibiot. 1991, 44, 903−907. (e) Tanaka, M.; Shin-ya, K.; Furihata, K.; Seto, H. J. Antibiot. 1995, 48, 326−328. (f) Roy, M. K.; Thalang, V. N.; Trakoontivakorn, G.; Nakahara, K. Biochem. Pharmacol. 2004, 67, 41−51. (5) (a) Brooker, R.; Gajjar, K. T. Curr. Sci. 1953, 22, 44−45. (b) Kirtikar, K. R.; Basu, B. D. Ind. Med. Plants 1960, 1, 473. (6) (a) Narasimhan, N. S.; Paradhar, M. V.; Chitguppi, V. P. Tetrahedron Lett. 1968, 53, 5501−5504. (b) Chowdhury, B. K.; Chakraborty, D. P. Phytochemistry 1971, 10, 1967−1970. (c) Chakraborty, D. P.; Roy, S.; Ruha, R. J. Indian Chem. Soc. 1978, 55, 1114− 1115. (d) Rao, R. A. V.; Rhide, K. S.; Mujumdar, R. B. Chem. Ind. 1980, 17, 697−698. (e) Bhattacharyya, L.; Roy, S. K.; Chakraborty, D. P. Phytochemistry 1982, 21, 2432−2433. (f) Roy, S.; Bhattacharyya, L.; Roy, S. K.; Chakraborty, D. P. J. Indian Chem. Soc. 1982, 59, 1369− 1371. (g) Bhattacharyya, P.; Chowdhury, B. K. Indian J. Chem. 1985, 24B, 452−452. (7) (a) Ito, C.; Thoyama, Y.; Omura, M.; Kajiura, I.; Furukawa, H. Chem. Pharm. Bull. 1993, 41, 2096−2100. (b) Reisch, J.; Adebajo, A. C.; Kumar, V.; Aladesanmi, A. J. Phytochemistry 1994, 36, 1073−1076. (c) Saha, C.; Chowdhury, B. K. Phytochemistry 1998, 48, 363−366. (d) Nutan, M. T. H.; Hasan, C. M.; Rashid, M. A. Fitoterapia 1999, 70, 130−133. (e) Knolker, H. J.; Reddy, K. R. Chem. Rev. 2002, 102, 4303−4427. (f) Adebajo, A. C.; Reisch, J. Fitoterapia 2000, 71, 334− 337. (g) Narasimhan, N. S.; Paradkar, M. V.; Kelkar, S. L. Indian J. Chem. 1970, 8, 473−474. (h) Narasimhan, N. S.; Paradkar, M. V.; Chitguppi, V. P.; Kelkar, S. L. Indian J. Chem. 1975, 13, 993−999. (8) (a) Roglic, G.; Unwin, N. Diabetes Res. Clin. Pract. 2010, 87, 15− 19. (b) Kordik, C. P.; Reitz, A. B. J. Med. Chem. 1991, 42, 181. G



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(21) Quenching data were analyzed according to the following Stern−Volmer equation: I0/I = Kq[Q] + 1, where I0 is the emission intensity in the absence of a quencher, I is the emission intensity in the presence of a quencher, Kq is the quenching constant, and [Q] is the quencher concentration. The Kq value is obtained as a slope from the plot of I0/I versus [Q]. Further the apparent binding constant (Kapp) values were obtained for the compounds using the following equation: KEB[EB] = Kapp[compound], where the compound concentration has the value at a 50% reduction of the fluorescence intensity of EB, KEB = 1.0 × 107 M−1, and [EB] = 5 μM. (22) Shahabuddin, M. S.; Gopal, M.; Raghavan, S. C. J. Cancer Mol. 2007, 3, 139−146. (23) (a) Bailly, C.; Qu, X.; Chaires, J. B.; Colson, P.; Houssier, C.; Ohkubo, M.; Nishimure, S.; Yoshinari, T. J. Med. Chem. 1999, 42, 2927−2935. (b) Tanious, F. A.; Ding, D.; Patrick, D. A.; Tidwell, R. R.; Wilson, W. D. Biochemistry 1997, 36, 15315−15325. (24) Raja, D. S.; Bhuvanesh, N. S. P.; Natarajan, K. Inorg. Chem. 2011, 50, 12852−12866. (25) The equilibrium binding constant (Kbin) can be analyzed according to the Scatchard equation: log[(I0 − I)/I] = log Kbin + n log[Q]. From the plot of log(I0 − I)/I versus log[Q], the number of binding sites (n) and the binding constant (Kbin) have been obtained. (26) Raja, D. S.; Paramaguru, G.; Bhuvanesh, N. S. P.; Reibenspies, J. H.; Renganathan, R.; Natarajan, K. Dalton Trans. 2011, 40, 4548− 4559. (27) Hu, Y.; Yang, Y.; Dai, C.; Liu, Y.; Xiao, X. Biomacromolecules 2010, 11, 106−112.

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Bioactive Dimeric Carbazole Alkaloids from Murraya koenigii

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