Two acridones and two coumarins from the roots of Paramignya trimera

Accepted Manuscript Two acridones and two coumarins from the roots of Paramignya trimera Phu H. Dang, Tho H. Le, Kim-Phuong T. Phan, Tuyet-Phuong T. Le, ThanhMai T. Nguyen, Nhan T. Nguyen PII: DOI: Reference:

S0040-4039(17)30269-1 http://dx.doi.org/10.1016/j.tetlet.2017.02.083 TETL 48694

To appear in:

Tetrahedron Letters

Received Date: Revised Date: Accepted Date:

23 January 2017 24 February 2017 27 February 2017

Please cite this article as: Dang, P.H., Le, T.H., Phan, K.T., Le, T.T., Nguyen, T.T., Nguyen, N.T., Two acridones and two coumarins from the roots of Paramignya trimera, Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/ j.tetlet.2017.02.083

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract

Two acridones and two coumarins from the roots of Paramignya trimera

Leave this area blank for abstract info.

Phu H. Dang, Tho H. Le, Kim-Phuong T. Phan, Tuyet-Phuong T. Le, Thanh-Mai T. Nguyen, Nhan T. Nguyen

1

Tetrahedron Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m

Two acridones and two coumarins from the roots of Paramignya trimera Phu H. Dang a, Tho H. Le a, Kim-Phuong T. Phan a, Tuyet-Phuong T. Le a, Thanh-Mai T. Nguyen a,b, Nhan T. Nguyen a,b,* a b

Faculty of Chemistry, VNUHCM–University of Science, 227 Nguyen Van Cu, Ho Chi Minh City, Vietnam Cancer Research Laboratory, Vietnam National University Ho Chi Minh City, 227 Nguyen Van Cu, Ho Chi Minh City, Vietnam

A R T IC LE IN F O

A B S TR A C T

Article history: Received Received in revised form Accepted Available online

Two acridones, paratrimerins C (1) and D (2), and two coumarins, paratrimerins E (3) and F (4), were isolated from the CHCl3 and EtOAc extracts of Paramignya trimera (Rutaceae), together with twelve known compounds (5–16). Their structures were elucidated on the basis of spectroscopic data. All isolated compounds possessed significant α-glucosidase inhibitory activity in a concentration-dependent manner, and showed more potent inhibitory activity, with IC50 values ranging from 14.6 to 112.2 µM, than the positive control acarbose (IC50, 214.5 µM). The biosynthesis of the isolated coumarins and acridones was proposed.

Keywords: Paramignya trimera Rutaceae Acridone Coumarin α-glucosidase

Introduction Paramignya trimera (Oliv.) Guillaum is a woody shrub belonging to the family Rutaceae, which is found in Thailand and Vietnam. P. trimera, local name “Xao tam phan”, is an endemic plant in South Vietnam. The roots of this plant have been used as Vietnamese traditional medicines for the treatment of diabetes.1 Previously, two new coumarins, paratrimerins A and B, together with three known coumarins were isolated from roots and stems of P. trimera.2 As part of our continued study on the screening of medicinal plants for α-glucosidase inhibitory activity,3–6 we also found that the methanolic extract of the roots of P. trimera exhibited significant α-glucosidase inhibitory activity with an IC50 value of 36.6 µg/mL. Therefore, further phytochemical investigation of the roots of P. trimera collected from Khanh Hoa province was carried out; sixteen compounds including two new acridones and two new coumarins were isolated. Additionally, the α-glucosidase inhibitory activity of the isolated compounds was examined. Results and discussion The powdered roots of P. trimera were heated at reflux in MeOH. The MeOH extract was successively partitioned to yield petroleum ether, CHCl 3, EtOAc, and remaining aqueous fractions, respectively. Further separation and purification of the CHCl3 and EtOAc fractions led to the isolation of sixteen compounds: paratrimerins C−F (1−4), ostruthin (5),2 umbelliferone (6),7 scopoletin (7),8 ninhvanin (8),2 xanthyletin (9),9 pandanusin A (10),10 citrusinine-I (11),11 glycocitrine-III (12),12 oriciacridone E (13),13 5-hydroxynoracronycin (14),9 daedalin A (15),14 and vanillic acid (16).15

2016 Elsevier Ltd. All rights reserved.

Compound 1, paratrimerin C, showed the molecular formula C16H11NO4, as deduced from the positive HR-FAB-MS at m/z 282.0772 [M + H]+ (calcd for C16H12NO4, 282.0766). The IR spectrum exhibited absorption bands at 3435 cm−1 (O‒H) and 1660 cm−1 (C=O). In the 1H NMR spectrum (Table 1), the hydroxy group was situated peri to the carbonyl group in the 9acridone skeleton at δH 14.84 (s). The ABC pattern signals at δH 7.64 (dd, J = 7.8, 1.6 Hz), 7.28 (dd, J = 7.8, 1.6 Hz), and 7.21 (t, J = 7.8 Hz) due to H-8, H-6 and H-7, respectively, indicated that the A ring was trisubstituted. Moreover, the three-protons singlet at δH 4.24 (s) confirmed the N-methyl group in the 10-methyl-9acridone skeleton. Three signals at δH 7.94 (d, J = 2.3 Hz, H-2′), 7.47 (dd, J = 2.3, 0.9 Hz, H-1′), and 6.80 (d, J = 0.9 Hz, H-2) coupled to each other, and the long-range coupling ( 5J = 0.9 Hz) between signals at δH 7.47 and 6.80 assigned to H-1′ and H-2, respectively, suggested the presence of the 4,5,6-trisubstituted benzofuran moiety in 1. 16 The 13C NMR spectrum (Table 1) showed the presence of fourteen aromatic carbons (δC 91.6– 160.5), one carbonyl (δC 181.4), and one downfield-shifted Nmethyl (δC 45.5). The angular orientation of 1 was confirmed by the 13C NMR data of three methine carbons of the benzofuran moiety (δC 91.6, 108.5, 142.7) in comparison to those reported in the literature.16 The 1- and 5-OH were confirmed by the HMBC correlations between H-6/C-5, H-7/C-5, H-2/C-1, and 1-OH/C-1. The HMBC correlations between H-1′/C-3, H-1′/C-4, H-2′/C-3, and H-2′/C-4 also indicated the presence of the benzofuran moiety (Fig. 2). Due to the small isolated amount of 1, it was not possible to record NOE enhancements. Thus, the structure of paratrimerin C was concluded as 1.

2

Tetrahedron Letters

Figure 1. Structures of compounds 1–16.

Compound 2, paratrimerin D, showed the molecular formula C24H27NO5, as deduced from the positive HR-FAB-MS at m/z 410.1986 [M + H]+ (calcd for C24 H28NO5, 410.1967). The IR spectrum exhibited absorption bands at 3445 cm−1 (O‒H) and 1665 cm−1 (C=O). The 1H NMR spectrum (Table 1) showed the peri-hydroxycarbonyl signal at δH 16.41 (s), and N-methyl at δH 3.90 (s) in the 10-methyl-9-acridone skeleton. Three aromatic signals at δH 6.99 (2H, m, H-6, and -7) and 7.76 (1H, m, H-8) indicated the trisubstituted A ring. Moreover, the 1H NMR spectrum also showed a singlet aromatic signal at δH 6.32, indicating the presence of a pentasubstituted C ring. Three tertiary methyls (δH 1.44, 1.41, 1.40), two methines (δH 3.74, 1.75), and three methylenes (δH 1.39–2.11) suggested the presence of the cyclized geranyl moiety. The 13C NMR spectrum (Table 1) showed the presence of twelve aromatic carbons (δC 91.5–163.8), one carbonyl (δC 180.2), one N-methyl (δC 40.6), three methyls (δC 25.9, 28.8, 30.2), three methylenes (δC 21.5, 38.7, 40.0), two methines (δC 26.4, 53.1), and two oxygenated tertiary carbons (δC 72.6, 76.6). The 1H-1H COSY correlations between H-1′/H-6′, H-1′/H-2′, H-6′/H-5′ indicated a 9oxahydrophenanthrene-type moiety in 2. This conclusion was confirmed by HMBC correlations between H-4/C-3, H-1′/C-2, H1′/C-2′, H-6′/C-2, H-6′/C-5′, H-1′/C-6′, and H-6′/C-1′. The HMBC correlations between Me-9′/C-3′, Me-8′/C-7′, and Me10′/C-7′ suggested these methyl groups were attached to the corresponding oxygenated tertiary carbon. The singlet signal at δH 6.32 assigned to H-4 of the B ring in the acridone skeleton, was confirmed by the HMBC correlations between N-CH3/C-4a, and H-4/C-4a. The 5-OH was confirmed by the HMBC correlations between H-6/C-5, and H-7/C-5 (Fig. 2). The NOESY correlation between H-4/N-CH3 verified the linear pyrano[3,2b]acridone. Moreover, NOESY correlations between H-1′/H-6′, H-1′/H-2′a, H-1′/H-2′e, H-6′/Me-10′, Me-9′/H-2′a, and Me-9′/H2′e suggested the relative structure of 2 (Fig. 2). Consequently, the above spectral data afforded the structure of paratrimerin D as 2.

Table 1. 1H (500 MHz) and compounds 1 and 2 Position

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 10-NCH3 1-OH 1′ 2′

13

C (125 MHz) NMR data for

Paratrimerin C (1a) δH δC (J in Hz) 160.5 – 91.6 6.80, d (0.9) 159.3 – 107.0 – 141.8 – 150.6 – 120.0 7.28, dd (7.8, 1.6) 123.8 7.21, t (7.8) 112.9 7.64, dd (7.8, 1.6) 123.7 – 181.4 – 106.5 – 135.2 – 45.5 4.24, s 14.86, s 108.5 7.47, dd (2.3, 0.9) 142.7 1.05, d (2.3)

Paratrimerin D (2b) δH δC (J in Hz) 160.4 – 105.3 – 163.8 – 91.5 6.32, s 145.6 – 145.6 – 120.3 6.99, m 122.0 118.1 123.0 180.2 104.2 133.4 40.6 26.4 40.0

3′ 4′

– –

– –

76.6 38.7

5′

–

–

21.5

6′

–

–

53.1

7′ – – 8′ – – 9′ – – 10′ – – a b δ values in DMSO-d6; δ values in CDCl 3

72.6 25.9 28.8 30.2

6.99, m 7.76, m – – – – 3.90, s 16.41, s 3.74, m 1.93, dd (13.1, 2.8) 1.82, ddd (13.1, 2.9, 2.9) – 2.11, m 1.63, m 1.61, m 1.39, m 1.75, ddd (12.8, 2.9, 2.9) – 1.41, s 1.44, s 1.00, s

3 9'

O 8

OH

OH

8

7'

HO

OH 1

9'

10'

3'

O

1

1'

5'

3' 1'

6

6' 3

6'

5'

6

N

4

N

5

OH

1'

1

OH

7'

O

4

2'

HO

8'

O

O

7

O

O 9'

3

10'

OH

7

3

2' 3'

5

4

1'

4

7' 8'

10'

8'

O

O

4

2

H

HO H

H H

H

H N

H

H

H

O OH

O O

O

O

HO 4 2

Figure 2. Connectivities (bold lines) deduced by the COSY, significant HMBC (solid arrows) and NOESY (dashed arrows) correlations observed for 1–4

Compound 3, paratrimerin E, showed the molecular formula C19H22O4, as deduced from the positive HR-ESI-MS at m/z 355.1527 [M + Na]+ (calcd for C19H22 O4Na, 355.1521). The IR spectrum exhibited absorption bands at 3310 cm−1 (O–H) and 1720 cm−1 (C=O). The 1H NMR spectrum (Table 2) showed characteristic signals similar to those of ostruthin,2 with two cisolefinic protons at δH 6.21 (d, J = 9.4 Hz, H-3), 7.61 (d, J = 9.4 Hz, H-4), and two singlet aromatic protons at δH 7.18 (s, H-5), 6.84 (s, H-8) indicated the 6,7-disubstituted coumarin. Moreover, the presence of an oxymethine proton at δH 3.40 (m), and two upfield-shifted methyls at δH 1.21 (s), 1.17 (s) suggested that the modified geranyl side-chain was attached to C-6. The 13C NMR spectrum (Table 2) displayed the presence of six aromatic carbons (δC 103.5–158.5), four olefinic carbons (δC 112.9, 122.0, 138.4, 143.9), one carbonyl (δC 162.2), three methylenes (δC 29.2, 29.5, 37.0), one oxymethine (δC 78.3), one oxygenated tertiary carbon (δC 73.4), and three methyls (δC 16.4, 23.4, 26.6). The HMBC correlations from Me-8′ and Me′-10 to the oxygenated tertiary and secondary carbons at δC 73.4 and 78.3, respectively, indicated that the ∆6′ of geranyl side-chain was dihydroxylated. The 6,7-disubstituted coumarin was also confirmed by the HMBC correlations between H-1′/C-5, and H1′/C-7 (Fig. 2). A NOESY spectrum was not recorded due to the small isolated amount of 3. Thus, the structure of paratrimerin E was defined as 3. Compound 4, paratrimerin F, showed the molecular formula C19H20O3, as deduced from the positive HR-ESI-MS at m/z 297.1467 [M + H]+ (calcd for C19H21O3, 297.1491). The IR spectrum exhibited absorption bands at 3300 cm−1 (O‒H) and 1725 cm−1 (C=O). The 1H NMR spectrum (Table 2) showed characteristic signals of the 6,7-disubstituted coumarin, with two cis-olefinic protons at δH 6.23 (d, J = 9.4 Hz, H-3) and 7.58 (d, J = 9.4 Hz, H-4), and two singlet aromatic protons at δH 6.97 (s, H5), 6.82 (s, H-8). Three tertiary methyls (δH 1.39, 1.38, 0.69), three methines (δH 3.09, 2.69, 2.46), and two methylenes (δH 1.65–1.91) suggested the presence of the cyclized geranyl sidechain. The 13C NMR spectrum (Table 2) showed the presence of six aromatic carbons (δC 106.2–154.4), two olefinic carbons (δC 113.5, 143.7), one carbonyl (δC 161.9), three methines (δC 39.3, 39.7, 46.8), two methylenes (δC 25.6, 39.0), one oxygenated tertiary carbon (δC 85.4), one quaternary carbon (δC 39.4), and

three methyls (δC 19.6, 26.9, 34.9). The HMBC correlations between H-1′/C-3′, H-2′/Me-9′, Me-9′/C-2′, Me-9′/C-3′, and Me9′/C-7 suggested the presence of the 2-methyl-3,4-dihydro-2Hpyran ring. The cyclopentane-fused ring was indicated by the HMBC correlations between H-4′/C-3′, H-5′/C-3′, H-6′/C-2′, and H-6′/C-3′. Moreover, the HMBC correlations between H-1′/C-7′, H-1′/C-8′, H-1′/C-10′, Me-8′/C-6′, and Me-10′/C-6′ indicated the presence of the 1,1-dimethylcyclobutane ring (Fig. 2). Based on these NMR data, the structure of 4 was concluded as 5dehydroxyeriobrucinol.17 The NOESY correlations between H4/H-5, H-5/H-1′, H-5/Me-10′, H-1′/H-2′, H-1′/Me-8′, H-2′/Me-9′, H-2′/H-6′, and H-6′/Me-8′ indicated the all cis arrangement of these protons corresponding to a half chair conformation for the dihydropyran ring.18 Thus, the stereostructure of 4 was suggested as shown in Figure 2. All these data were in full agreement with the structure for paratrimerin F.

Table 2. 1H (500 MHz) and compounds 3 and 4 in CDCl3 Position 2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′

Paratrimerin E (3) δH δC (J in Hz) 162.2 – 112.9 6.21, d (9.4) 143.9 7.61, d (9.4) 112.9 – 128.7 7.18, s 125.3 – 158.5 – 103.5 6.84, s 154.5 – 29.2 3.39, m 122.0 5.37, t (7.2) 138.4 – 37.0 2.32, m 2.19, m 29.5 1.63, m 1.48, m 78.3 3.40, m 73.4 – 26.6 1.21, s 16.4 1.77, s 23.4 1.17, s

13

C (125 MHz) NMR data for

δC 161.9 113.5 143.7 113.5 128.3 122.2 158.0 106.2 154.4 39.7 39.3 85.4 25.6 39.0 46.8 39.4 34.9 26.9 19.6

Paratrimerin F(4) δH (J in Hz) – 6.23, d (9.4) 7.58, d (9.4) – 6.97, s – – 6.82, s – 3.09, d (9.6) 2.69, dd (9.6, 7.9) – 1.70, m 1.91, m 1.65, m 2.46, ddd (7.9, 7.9, 3.0) – 1.38, s 1.39, s 0.69, s

4

Tetrahedron Letters

Scheme 1. Plausible biosynthetic pathways for 1–14

All isolated compounds were tested for their α-glucosidase inhibitory activity according to the previous procedure.4 Acarbose, which is currently used clinically in combination with either diet or antidiabetic agents to control the blood glucose level of patients, was used as the positive control in this study. All compounds (1–16) possessed significant α-glucosidase inhibitory activity (Table 3). Both paratrimerin C (1) and oriciacridone E (14) had the most potent inhibitory activity, with an IC50 value of 14.6 µM; these compounds were 15 times more active than acarbose (IC50, 214.5 µM). The cyclization of a prenyl or a geranyl moiety decreases the inhibition activity (5 > 4 > 10,

13 > 2, 14 > 15). The methoxy group also causes a decrease in activity (5 >> 8, 6 > 7). The ∆6′ dihydroxylation of the geranyl moiety also decreases the activity (3 >> 5). The coumarin and acridone were major constituents of the Rutaceae family. Thus, we have proposed plausible biogenetic pathways for the isolated coumarins and acridones from the simple co-metabolites, umbelliferone (6) and 1,3,5-trihydroxy9(10H)-acridone (17), respectively (Scheme 1). NADPH oxidase generates superoxide by transferring electrons from NADPH to molecular oxygen to produce the superoxide anion, subsequently causing the monooxygenase reaction.19 The methylation reaction

5 was catalyzed by methyltransferase with S-adenosyl methionine (SAM) as a co-substrate.20 Ostruthin (5), the main component of P. trimera,21 was biosynthesized from 6 by Orf2 in the presence of Mg2+.22 The oxidative bond cleavage of 14 that leads to paratrimerin C (1) was catalyzed by cytochrome P450 requiring O2 and NADPH as a co-factor.23

Table 3. α-Glucosidase inhibitory activity of the isolated compounds Compound

a

IC50 (µM)

Compound

IC50 (µM)

Compound

IC50 (µM)

1

14.6

7

69.0

13

45.5

2

70.0

8

84.6

14

14.6

3

106.9

9

37.5

15

63.9

4

31.7

10

95.3

16

49.4

5

17.1

11

112.2

acarbosea

214.5

6

32.4

12

81.8

postive control

Conclusions

From the CHCl3 extract of P. trimera, two new acridones and two new coumarins were isolated together with twelve known compounds. All compounds showed significant αglucosidase inhibitory activity. These results suggested that the traditional use of P. trimera for the treatment of diabetes in Vienam may be attributable to the α-glucosidase inhibitory activity of its acridones and coumarins. We also proposed the plausible biogenetic pathways for the formation of 1–14. Acknowledgements This research was supported by a grant from the Vietnam National University Ho Chi Minh City (No. A2015-18-02) to M. T. T. N. Supplementary data Supplementary data (experimental, NMR, and HRMS spectra of 1–4) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet. References and notes 1. Pham, H. H. In An illustrated flora of Vietnam; Tre Publishing House, Ho Chi Minh City, 1999; Vol. 2. pp 439.

2. Cuong, N. M.; Huong, T. T.; Khanh, P. N.; Tai, N. V.; Ha, V. T.; Son, N. T.; Tai, B. H.; Kim, Y. H. Chem. Pharm. Bull. 2015, 63, 945–949. 3. Nguyen, H. X.; Le, T. C.; Do, T. N.; Le, T. H.; Nguyen, N. T.; Nguyen, M. T. Chem. Cent. J. 2016, 10, 45–50. 4. Dang, P. H.; Nguyen, N. T.; Nguyen, H. X.; Nguyen, L. B.; Le, T. H.; Do, T. N. V.; Can, M. V.; Nguyen, M. T. T. Fitoterapia 2015, 100, 201–207. 5. Dang, P. H.; Nguyen, H. X.; Nguyen, N. T.; Le, H. N. T.; Nguyen, M. T. T. Phytother. Res. 2014, 28, 1632–1636. 6. Nguyen, M. T. T.; Nguyen, N. T.; Nguyen, H. X.; Huynh, T. N. N.; Min, B. S. Nat. Prod. Sci. 2012, 18, 47–51. 7. Kutubi, M. S.; Hashimoto, T.; Kitamura, T. Synthesis 2011, 2011, 1283–1289. 8. Fei, D.-Q.; Arfan, M.; Rafiq, J.; Gao, K. Chem. Nat. Compd. 2009, 45, 896–897. 9. Wu, T.; Kuoh, C.; Furukawa, H. Chem. Pharm. Bull. 1983, 31, 895–900. 10. Nguyen, T. P.; Le, T. D.; Minh, P. N.; Dat, B. T.; Pham, N. K. T.; Do, T. M. L.; Nguyen, D. T.; Mai, T. D. Nat. Prod. Res. 2016, 30, 2389–2395. 11. Kato, N.; Fujita, M.; Fujimura, K.-i.; Kawashima, Y.; Nishiyama, Y. Chem. Pharm. Bull. 1993, 41, 445-452. 12. Ito, C.; Kondo, Y.; Rao, K. S.; Tokuda, H.; Nishino, H.; Furukawa, H. Chem. Pharm. Bull. 1999, 47, 1579–1581. 13. Wansi, J. D.; Wandji, J.; Mbaze Meva'a, L.; Kamdem Waffo, A. F.; Ranjit, R.; Khan, S. N.; Asma, A.; Iqbal, C. M.; Lallemand, M.-C.; Tillequin, F.; Fomum Tanee, Z. Chem. Pharm. Bull. 2006, 54, 292–296. 14. Morimura, K.; Hiramatsu, K.; Yamazaki, C.; Hattori, Y.; Makabe, H.; Hirota, M. Biosci., Biotechnol., Biochem. 2009, 73, 627–632. 15. Feng, W.-S.; Li, K.-K.; Zheng, X.-K. Acta Pharmaceutica Sinica B 2011, 1, 36–39. 16. Reisch, J.; Mester, I.; Kapoor, S. K.; Rózsa, Z.; Szendrei, K. Liebigs Ann. Chem. 1981, 1981, 85–91. 17. Jefferies, P. R.; Worth, G. K. Tetrahedron 1973, 29, 903–908. 18. Rashid, M. A.; Armstrong, J. A.; Gray, A. I.; Waterman, P. G. Phytochemistry 1992, 31, 3583–3588. 19. Paravicini, T. M.; Touyz, R. M. Diabetes Care 2008, 31, S170– S180. 20. Struck, A.-W.; Thompson, M. L.; Wong, L. S.; Micklefield, J. ChemBioChem 2012, 13, 2642–2655. 21. Nguyen, V. T.; Bowyer, M. C.; Vuong, Q. V.; Altena, I. A. V.; Scarlett, C. J. Industrial Crops and Products 2015, 67, 192–200. 22. Kuzuyama, T.; Noel, J. P.; Richard, S. B. Nature 2005, 435, 983–987. 23. Stanjek, V.; Miksch, M.; Lueer, P.; Matern, U.; Boland, W. Angew. Chem. Int. Ed. 1999, 38, 400–402.