Bioorganic & Medicinal Chemistry Letters 24 (2014) 5656–5659

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Pyrrole alkaloids from the fruits of Morus alba Seon Beom Kim a, Bo Yoon Chang b, Bang Yeon Hwang a, Sung Yeon Kim b,⇑, Mi Kyeong Lee a,⇑ a b

College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 362-763, Republic of Korea College of Pharmacy, Wonkwang University, Iksan, Chonbuk 570-749, Republic of Korea

a r t i c l e

i n f o

Article history: Received 15 September 2014 Revised 21 October 2014 Accepted 23 October 2014 Available online 29 October 2014 Keywords: Morus alba Pyrrole alkaloids Morroles B–F Pancreatic lipase Moraceae Mulberry

a b s t r a c t Phytochemical investigation of the fruits of Morus alba afforded seventeen pyrrole alkaloids including five new compounds. The structures of five new pyrrole alkaloids, named morroles B–F (4, 5, 7, 16 and 17), were determined on the basis of spectroscopic interpretations. 4-[Formyl-5-(hydroxymethyl)-1Hpyrrol-1-yl]butanoate (2) was synthesized by chemical reaction but first isolated from nature. Among isolated compounds, compounds 6 and 14 significantly inhibited pancreatic lipase activity. Ó 2014 Elsevier Ltd. All rights reserved.

Morus alba, which belongs to Moraceae family, is a deciduous tree that is widely distributed in Asia. All parts of this tree such as roots, leaves and fruits have been used in traditional medicine.1 Among them, the fruits of M. alba, commonly called mulberry, are generally consumed as fresh fruits and also consumed as juices and jams. Alcoholic beverages like wine and vinegar are also available. In traditional medicine, mulberry is used as a general tonic and an immunostimulant for health and longevity. Recent studies also suggested its antioxidant, anti-inflammatory, anti-diabetic and memory-enhancing effects.2–4 Mulberry is rich in diverse phenolic compounds such as anthocyanins, flavonoids and polyphenols.5–7 In our previous study, we isolated pyrrole alkaloids from M. alba fruits for the first time and suggested their immunostimulatory activity.8 We have further tried to investigate pyrrole alkaloids from M. alba fruits and our intensive study resulted in the isolation of seventeen pyrrole alkaloids including five new compounds from M. alba fruits (Fig. 1).9 In this Letter, the isolation and structure elucidation of five new pyrrole alkaloids, morroles B–F, are described. The inhibitory effect on pancreatic lipase activity was also investigated. Compound 4 was obtained as a brown gum and a molecular formula of C9H11NO4 was determined on the basis of HRESIMS at m/z 196.0616 [MH] (calcd 196.0610). The maximum UV absorption ⇑ Corresponding authors. Tel.: +82 63 850 6768; fax: +82 63 857 0342 (S.Y.K.); tel.: +82 43 261 2818; fax: +82 43 268 2732 (M.K.L.). E-mail addresses: [email protected] (S.Y. Kim), [email protected] (M.K. Lee). http://dx.doi.org/10.1016/j.bmcl.2014.10.073 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

was observed at 293 nm, which is characteristic for pyrrole-2-aldehyde.8 The presence of pyrrole-2-aldehyde was also supported from signals at dH 6.16 (1H, d, J = 4.0 Hz), dH 6.88 (1H, d, J = 4.0 Hz), and dH 9.41 (1H, s) in the 1H NMR spectrum. The 1H and 13C NMR spectra of 4 also indicated the presence of a hydroxymethyl group [dH 4.38 (1H, d, J = 14.0 Hz), 4.44 (1H, d, J = 14.0 Hz), and dC 55.9]. Additionally, one secondary methyl signals at [dH 1.51 (3H, d, J = 7.2 Hz) and dC 19.9], one methine signals at [dH 5.62 (1H, br s) and dC 56.0] and one carbonyl signal at dC 174.4 were observed in the 1H, 13C NMR and HSQC spectra. The HMBC correlations between aldehyde signal at dH 9.41 (H-1) and dC 132.1 (C-2), dC 123.4 (C-3), and between dH 4.38, 4.44 (H-6) and dC 110.3 (C-4), 132.1 (C-5) suggested that the aldehyde and hydroxymethyl moieties were connected to C-2 and C-5 of the pyrrole ring, respectively. The HMBC correlations between dH 1.51 (H-30 ) and dC 174.4 (C-10 ), dC 56.0 (C-20 ) as well as between dH 5.62 (H-20 ) and dC 174.4 (C-10 ) suggested the presence of CH3–CH–COOH. The linkage of CH3–CH–COOH to the pyrrole moiety was deduced to be nitrogen of the pyrrole ring by the correlation between dH 5.62 (H-20 ) and dC 132.1 (C-2) in the HMBC spectrum (Fig. 2). On the basis of the obtained data, compound 4 was determined as 2-(2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl)propanoic acid and named morrole B.10 Compound 5 was purified as a brown gum with a molecular formula of C10H13NO4 (HRESIMS m/z 234.0740 [M+Na]+, calcd 234.0742). The 1H and 13C NMR spectra of 5 differ from those of 4, only for the presence of additional methoxyl signals at [dH 3.24 (3H, s) and dC 57.7]. The position of additional methoxyl

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S. B. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5656–5659

OH

O

O 3'

R 2O

O

O

N

H

H

OR 1

2'

N

2

1

1' 5

3

1 R 1 =CH3 R2 =H 2 R 1 =H R2 =CH 3 3 R 1 =CH3 R2 =CH 3

OR2

H 2N

6

OR 1

O

4'

O

OCH 3

N

H

4 R 1 =H R2 =H 5 R 1 =CH3 R2 =H 6 R 1 =CH3 R2 =CH 3

1'

2'

O

4

O

3'

OCH3

O

OCH 3

N

H

8

7

OCH 3

R

O O H

R2

O O

O

O

N

R1 O

N

H

O

O

O N

H

O H

10

9

11 R 1=H R 2 =CH3 12 R 1=CH3 R2 =H

13 R=H 14 R=OH

O

O

5'

6'

NH

4'

O

O N

3'

2'

N

H

NH

5'

O

O

OR

4' 2'

3'

N

H

15 R=H 16 R=CH3

O OH

17

Figure 1. Chemical structures of compounds 1–17 isolated from M. alba fruits.

O O H

H 2N

O

OH N

O

OH

H



OH N

O

OCH 3

O

OCH 3

N

H

O

O

O

OCH 3





H

O

NH

NH O

N

O

O OCH 3



H

N

OH



Figure 2. HMBC correlations of compounds 4, 5, 7, 16 and 17.

moiety was confirmed to C-6 by the correlation between dH 3.24 (OCH3) and dC 65.4 (C-6) in the HMBC spectrum (Fig. 2). Thus, compound 5 was established as a methoxyl derivative of 4 and named morrole C.11 Compound 7 was isolated as a brown gum and a molecular formula of C12H16N2O5 was determined from HRESIMS at m/z 291.0951 [M+Na]+ (calcd 291.0957). The 1H NMR spectrum of 7 was similar to those of 6 except for the replacement of a methyl signal at dH 1.55 in 6 by signals for methylene [dH 2.70 (1H, dd, J = 8.8, 16.4 Hz), dH 3.15 (1H, dd, J = 4.4, 16.4 Hz)] and NH2 [dH

6.87 (1H, s), dH 7.41 (1H, s)] in 7. Besides methylene signal at dC 37.9, additional carbonyl signal was observed at dC 171.4 in the 13 C NMR spectrum. The HMBC correlations between dH 2.70, 3.15 (H-30 ) and 55.5 (C-20 ), 171.4 (C-40 ) suggested the presence of H2N–CO–CH2–CH–COOH. On the basis of the obtained data, compound 7 was determined as methyl 4-amino-2-(2-formyl-5(methoxymethyl)-1H-pyrrol-1-yl)-4-oxobutanoate and named morrole E.12 Compound 16 was obtained as brown gum and a molecular formula of C11H12N2O4 was determined on the basis of HRESIMS

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S. B. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5656–5659

Table 1 H NMR data for compounds 4, 5, 7, 16 and 17 in DMSO-d6 (d in ppm, J in Hz)

1

H

4a

5a

1 3 4 6

9.41 s 6.88 (d 4.0) 6.16 (d 4.0) 4.38 (d 14.0) 4.44 (d 14.0) 5.62 br s 1.51 (d 7.2)

9.40 7.04 6.28 4.44 4.51 5.28 1.55

20 30

7a s (d (d (d (d br (d

9.37 7.11 6.28 4.36 4.79 5.60 2.70 3.15

4.0) 4.0) 12.8) 12.8) s 7.2)

16b s (d 4.0) (d 4.0) (d 12.8) (br d 12.8) br s (dd 8.8 16.4) (dd 4.4 16.4)

40

9.38 7.17 6.38 4.49

17a s (d 4.0) (d 4.0) s

5.32 (dd 5.6, 12.4)

2.64 (dd 7.0, 17.0) 3.03 (dd 9.0, 17.0)

2.08 2.39 2.59 2.83

5

a b

3.24 s

3.21 3.57 6.87 7.41

Table 2 C NMR data for compounds 4, 5, 7, 16 and 17 in DMSO-d6 (d in ppm)

a

m m m m

3.26 s

400 MHz. 500 MHz.

13

b

s s s s

s (d 4.0) (d 4.0) (d 5.2)

5.62 (t 7.0)

0

6-OCH3 10 -OCH3 40 -NH2

9.30 7.10 6.27 4.56

C

4a

5a

7a

16b

17a

1 2 3 4 5 6 10 20 30 40 50 60 6-OCH3 10 -OCH3

179.6 132.1 123.4 110.3 144.6 55.9 174.4 56.0 19.9

179.3 132.6 125.1 111.5 139.7 65.4 171.5 54.6 18.1

179.4 132.3 126.1 111.5 140.8 65.5 170.0 55.5 37.9 171.4

180.0 132.2 126.0 112.0 141.0 65.0

178.8 131.8 126.3 110.0 144.9 56.2

175.8 55.4 37.8 175.7

170.4 55.4 25.2 31.4 172.9

57.7

57.6 52.7

57.8

100 MHz. 125 MHz.

at m/z 259.0689 [M+Na]+ (calcd 259.0695). Comparison of 1H and 13 C NMR spectra of 16 with 15 showed that 16 differs from 15 only in the presence of additional methoxyl signals at [dH 3.26 (3H, s) and dC 57.8].13 The HMBC correlation between dH 3.26 (6-OCH3) and dC 65.0 (C-6) confirmed 16 as a methoxyl derivative of 15 and named morrole G.14 Compound 17 was isolated as a brown gum and a molecular formula of C11H12N2O4 was assigned on the basis of HRESIMS at m/z 259.0686 [M+Na]+ (calcd 259.0695). As similar to above

mentioned compounds, the signals at dH 6.27 (1H, d, J = 4.0 Hz), dH 7.10 (1H, d, J = 4.0 Hz), dH 9.30 (1H, s) and dH 4.56 (2H, d, J = 5.2 Hz) in the 1H NMR spectrum suggested the presence of a pyrrole-2-aldehyde-6-hydroxymethyl moiety. Besides the signals for a pyrrole-2-aldehyde-6-hydroxymethyl moiety, two methylene signals at [dH 2.08 (1H, m), 2.39 (1H, m), and dC 25.2] and [dH 2.59 (1H, m), 2.83 (1H, m), and dC 31.4], and one methine signals at [dH 5.32 (1H, dd, J = 5.6, 12.4 Hz) and dC 55.4] were observed in the 1H, 13 C and HSQC spectra. Additional two carbonyl carbon signals were also appeared at dC 170.4 and 172.9 in the 13C NMR spectrum. In the HMBC spectrum, correlations between dH 2.08, 2.39 (H-40 ) and dC 55.4 (C-30 ), 31.4 (C-50 ), 172.9 (C-60 ) and 170.4 (C-20 ), between dH 2.59, 2.83 (H-50 ) and dC 25.2 (C-40 ), 172.9 (C-60 ), and between dH 5.32 (H-30 ) and dC 25.2 (C-40 ), 170.4 (C-20 ), suggested the presence of a 2,6-dioxopiperidine moiety.15 The linkage of the dioxopiperidine to the nitrogen of the pyrrole ring was deduced from the correlations between dH 5.30 (H-30 ) and dC 131.8 (C-2), 144.9 (C-5) in HMBC spectrum (Fig. 2). Based on the obtained data, compound 17 was confirmed as 1-(2,6-dioxopiperidin-3-yl)-5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde and named morrole H.16 The known compounds were identified as 4-[formyl-5(hydroxymethyl)-1H-pyrrol-1-yl]butanoate (1),9 4-[formyl5-(hydroxymethyl)-1H-pyrrol-1-yl]butanoate (2),17 4-[formyl5-(methoxymethyl)-1H-pyrrol-1-yl]butanoic acid (3),9 methyl 2-[2-formyl-5-(methoxymethyl)-1H-pyrrole-1-yl]propanoate (6),18 methyl 2-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-3-

Figure 3. Effects of compounds 1–17 on pancreatic lipase activity. The activity was measured at 100 lM and expressed as mean ± SD of three independent experiments. ⁄ p