Phytochemistry xxx (2014) xxx–xxx

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Hispidacine, an unusual 8,40 -oxyneolignan-alkaloid with vasorelaxant activity, and hispiloscine, an antiproliferative phenanthroindolizidine alkaloid, from Ficus hispida Linn. Veronica Alicia Yap a, Bi-Juin Long b, Kang-Nee Ting b, Sandy Hwei-San Loh c, Kien-Thai Yong d, Yun-Yee Low e, Toh-Seok Kam e, Kuan-Hon Lim a,⇑ a

School of Pharmacy, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia Department of Biomedical Sciences, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia c School of Biosciences, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia d Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia e Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia b

a r t i c l e

i n f o

Article history: Received 13 September 2014 Received in revised form 24 October 2014 Available online xxxx Keywords: Ficus hispida Moraceae Alkaloids Neolignans Phenanthroindolizidine NMR Antiproliferation Vasorelaxation

a b s t r a c t Hispidacine, an 8,40 -oxyneolignan featuring incorporation of an unusual 2-hydroxyethylamine moiety at C-7, and hispiloscine, a phenanthroindolizidine alkaloid, were isolated from the stem-bark and leaves of the Malaysian Ficus hispida Linn. Their structures were established by spectroscopic analysis. Hispidacine induced a moderate vasorelaxant activity in rat isolated aorta, while hispiloscine showed appreciable antiproliferative activities against MDA-MB-231, MCF-7, A549, HCT-116 and MRC-5 cell lines. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The pantropical genus Ficus, which comprises ca. 735 species, of which ca. 99 occur in Peninsular Malaysia, is the largest of the 37 genera in the Moraceae family. Many plants of this genus are used in traditional medicines leading them to be the subject of phytochemical studies (Lansky et al., 2008). Ficus hispida Linn. is widely distributed in many parts of Asia (Sri Lanka, India, South China, Andaman Islands, and Malesia) as well as Australia (Queensland), and grows well in lowland forests including secondary forests (Berg and Corner, 2005). Various parts of this plant are traditionally used to treat illnesses such as ulcers, psoriasis, anemia, dysentery, piles, diabetes and jaundice (Ali and Chaudhary, 2011). Recent ethnopharmacological studies (in vitro and in vivo) of the plant extracts have showed additional therapeutic properties which include anticancer (Pratumvinit et al., 2009), cardioprotective ⇑ Corresponding author. Tel.: +60 3 89248208; fax: +60 3 89248018. E-mail address: [email protected] (K.-H. Lim).

(Shanmugarajan et al., 2008), hepatoprotective (Mandal et al., 2000), anti-diabetic (Ghosh et al., 2004) and nephroprotective (Swathi et al., 2011) activities. Despite the significant pharmacological activities shown by the crude extracts, there are limited reports on the biologically active metabolites from this plant. Previous phytochemical studies of F. hispida resulted in identification of phenanthroindolizidine alkaloids (Peraza-Sánchez et al., 2002; Venkatachalam and Mulchandani, 1982), glycosides (Asem and Laitonjam, 2008), sterols and terpenes (Ali and Chaudhary, 2011; Wang and Coviello, 1975). Although F. hispida is commonly available in Malaysia, none of the previously studied samples were collected from Malaysia. This led us to investigate the alkaloid composition of the stem-bark and leaves of the Malaysian sample, which yielded an unprecedented neolignan–alkaloid, as well as a new and a known phenanthroindolizidine alkaloid. Herein reported are the isolation and structure elucidation of the two new alkaloids (1 and 3, Fig. 1) as well as their vasorelaxant and cytotoxic activities.

http://dx.doi.org/10.1016/j.phytochem.2014.10.032 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Yap, V.A., et al. Hispidacine, an unusual 8,40 -oxyneolignan-alkaloid with vasorelaxant activity, and hispiloscine, an antiproliferative phenanthroindolizidine alkaloid, from Ficus hispida Linn. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.032

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V.A. Yap et al. / Phytochemistry xxx (2014) xxx–xxx

2. Results and discussion

OR3 2 '' 1''

Hispidacine (1) was obtained in minute amounts from the stem-bark of F. hispida as a colorless oil, [a]D +20 (c 0.28, CHCl3). Its IR spectrum showed absorption bands due to OH/NH functions (3385 cm1) and aromatic rings (1585 and 1505 cm1). The UV spectrum had absorption maxima at 228 and 271 nm, confirming the existence of aromatic moieties. The ESIMS of 1 from both an [M+H]+ ion at m/z 494 and HRESIMS measurements established the molecular formula as C25H35NO9, requiring 9° of unsaturation. The 13C NMR spectroscopic data of 1 (Table 1) indicated the presence of 19 discrete carbon signals, six of which are due to six pairs of equivalent carbons. The total number of carbon resonances in 1 was therefore 25 (comprising five methoxy, four methylene, two aliphatic methine, six aromatic/olefinic methine, six oxygenated aromatic methine and two quaternary aromatic carbon atoms), which is in agreement with the molecular formula. The six pairs of chemically equivalent resonances were due to two pairs of oxygenated aromatic carbons, two pairs of aromatic methine carbons and two pairs of aromatic methoxy groups. This, coupled with the presence of four other substituted aromatic carbons, suggested the presence of elements of symmetry in two tetrasubstituted aromatic rings, with each possessing a pair of equivalent aromatic methine and methoxy-substituted aromatic carbons. This was further supported by the presence of two 2-H aromatic singlets (d 6.57 and 6.61) and two 6-H methoxy singlets (d 3.81 and 3.82) in the 1H NMR spectroscopic data (Table 1). The above observations established the presence of two 1,3,4,5-tetrasubstituted aromatic rings with a plane of symmetry passing through C-1 and C-4 as well as C-10 and C-40 , in addition to requiring the pair of equivalent methoxy groups to be placed either at positions -3 and -5 or -2 and -6. The HMBC three-bond correlations from H-2/H-6 to C-7 and H20 /H-60 to C-70 confirmed the presence of the two methoxy pairs at C-3/C-5 and C-30 /C-50 , respectively (Fig. 2). The 1H NMR spectroscopic data also showed the presence of a rather deshielded methylene signal at d 4.30 (dd, J = 5.5, 1.6 Hz) and two olefinic resonances at d 6.28 (dt, J = 16, 5.5 Hz) and d 6.52 (dd, J = 16, 1.6 Hz), which indicated the presence of a 3hydroxypropenyl moiety corresponding to the Ar-C-70 –C-80 –C-90 – OH partial structure in 1. The large coupling constant observed between H-70 and H-80 (16 Hz) indicated the E geometry of the C-70 /C-80 double bond. This was also supported by NOESY data, which showed a correlation between H-70 and H-90 (Fig. 2). On the other hand, the 1H NMR signals at d 4.07 (d, J = 5 Hz), 4.11 (td, J = 5, 3 Hz), 3.64 (dd, J = 12, 3 Hz) and 3.91 (dd, J = 12, 5 Hz), with the COSY data, indicated the presence of a CHCH(O)CH2OH fragment, corresponding to the Ar-C-7–C-8–C-9–OH partial structure in 1 (Fig. 2). Additionally, the presence of a 2-hydroxyethylamine fragment corresponding to the NH–C-100 –C-200 –OH partial structure in 1 was also shown by the COSY data (Fig. 2). From the observations disclosed thus far, it is evident that 1 consists of

1 4

OMe

3

1

9

OMe

14a 4a 4b

3'

5'

7'

1 R1 = R 2 = R 3 = H 2 R1 = R 2 = R 3 = COCH 3

MeO

H

14

8a

O

13a

N

5

1'

MeO

O

4

8'

9

12 11

8 7

OMe

9'

OR 1

3

Fig. 1. Structures of 1–3.

Table 1 H and 13C NMR spectroscopic data of hispidacine (1) and the triacetate derivative (2).a

1

Position

1

2

dC

dH (J in Hz)

dC

dH (J in Hz)

1 2 3 4 5 6 7 8 9

135.19 104.80 153.35 137.15 153.35 104.80 64.56 86.64 61.52

– 6.57 – – – 6.57 4.07 4.11 3.64 3.91

135.30 104.88 153.24 137.10 153.24 104.88 63.93 82.89 63.17

– 6.60 – – – 6.60 3.88 4.56 4.29 4.51

10 20 30 40 50 60 70 80 90

133.13 103.61 153.41 135.18 153.41 103.61 130.60 128.84 63.44

– 6.61 – – – 6.61 6.52 6.28 4.30

132.21 103.80 153.47 134.65 153.47 103.80 134.08 122.96 64.92

– 6.65 s – – – 6.65 s 6.60 d (16) 6.24 dt (16, 6.5) 4.73 dd (6.5, 1.3)

100

49.78

42.26

2.65 m

200

61.96

64.67

4.16 m

3-OMe 4-OMe 5-OMe 30 -OMe 50 -OMe 3  OH/ 1  NH

56.18 60.90 56.18 56.24 56.24 –

2.57 ddd (12, 6, 3.8) 2.66 ddd (12, 6.9, 4) 3.64 ddd (10.5, 6, 4) 3.71 m 3.81 s 3.80 s 3.81 s 3.82 s 3.82 s 2.76 br s

56.10 60.78 56.10 56.11 56.11 –

3.84 s 3.83 s 3.84 s 3.87 s 3.87 s Not observed

9-OCOCH3

–

–

20.84b

1.94 sd

90 -OCOCH3

–

–

9 -OCOCH3 2 -OCOCH3 b–d

s d (5) td (5, 3) dd (12, 3) dd (12, 5) s

s dt (16, 1.6) dt (16, 5.5) dd (5.5, 1.6)

s

s m dt (7, 3) m dd (12, 7)

20.94b

2.08 sd

170.83c

0

200 -OCOCH3

s

170.75c

9-OCOCH3

a

OH

8S

O

MeO

00

OH

7S

MeO

OR2

HN MeO

–

–

20.98b

2.11 sd c

171.02

CDCl3, 600 MHz; assignments based on COSY, HSQC and HMBC. Assignments are interchangeable.

2'' 1''

HN

OMe

OMe

MeO

1 4

O

MeO

HN

OH

MeO

OH 8

9

OMe 3'

O

MeO OMe

MeO

7

1'

MeO

7'

5' 8'

(

= HMBC;

OH = NOESY)

9'

OH COSY correlations (bold bonds)

Fig. 2. Selected HMBC, NOESY and COSY correlations of 1.

two C6–C3 units and a 2-hydroxyethylamine moiety. Three-bond correlations from H-80 to C-10 , H-70 to C-20 /C-60 and H-20 /H-60 to C-70 in the HMBC spectrum confirmed the attachment of the 3hydroxypropenyl side-chain at C-10 , while correlations from H-20 / H-60 and H-8 to C-40 suggested attachment of C-8 to C-40 via an ether linkage. The C-7–C-8–C-9–OH fragment was deduced to be linked to the aromatic C-1 at C-7, from two- and three-bond correlations from H-7 to C-1, C-2/C-6 and H-2/H-6 to C-7. The threebond correlations from H-100 to C-7 and H-7 to C-100 indicated the attachment of the 2-hydroxyethylamine side chain to C-7. This

Please cite this article in press as: Yap, V.A., et al. Hispidacine, an unusual 8,40 -oxyneolignan-alkaloid with vasorelaxant activity, and hispiloscine, an antiproliferative phenanthroindolizidine alkaloid, from Ficus hispida Linn. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.032

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V.A. Yap et al. / Phytochemistry xxx (2014) xxx–xxx

leaves the remaining methoxy group which must be linked to C-4, which was confirmed by the observed three-bond correlations from 4-OMe to C-4 and H-2/H-6 to C-4. The proposed structure of hispidacine (1) is consistent with the HMBC and NOESY data (Fig. 2). The presence of three primary hydroxy groups in 1 was shown by acetylation, which yielded the triacetate derivative 2 (dH 1.94, 2.08 and 2.11 due to 3  COCH3; IR 1738 cm1; HRESIMS yielded the molecular formula C31H41NO12). The absence of the hydroxy groups in 2 was clearly indicated by the disappearance of the broad IR band at 3384 cm1 observed for 1, which was replaced instead by a weaker but sharper band at 3325 cm1 due to the secondary amine NH. The positions of the three hydroxy groups at C-9, C-90 and C-200 were further confirmed by the three-bond correlations from H-9, H-90 and H-200 to the respective acetyl carbons observed in the HMBC spectrum of 2. The two geminal hydrogens of C-100 , as well as C-200 in 1, unexpectedly gave rise to two pairs of non-equivalent signals (H-100 : d 2.57 and 2.66; H-200 : d 3.64 and 3.71), suggesting restricted C-100 –N bond rotation. This could be due to the occurrence of intramolecular hydrogen bonding between the hydroxy (H donor) and secondary amine (H acceptor) groups within the 2-hydroxyethylamine side-chain. Alternatively, the hydroxy group could form a hydrogen bond with the proximate aromatic methoxy group at C-30 /50 . This supposition was somewhat supported by the observation of two sets of equivalent geminal hydrogens due to H2C-100 and H2C-200 at d 2.65 and 4.16, respectively, in the 1H NMR spectroscopic data of the triacetate derivative 2 (Table 1). This suggested free rotation about the C-100 –N bond due to the absence of intramolecular hydrogen bonding following replacement of the hydroxy group at C-200 with an O-acetyl group. The structure of hispidacine (1) constitutes an 8,40 -oxyneolignan skeleton (Moss, 2000) featuring an unprecedented incorporation of a 2-hydroxyethylamine moiety at C-7. The relative configuration at the two stereocenters, C-7 and C-8, can be determined by analogy to the typical 8,40 -oxyneolignan system via examination of the magnitude of the coupling constant between H-7 and H-8. Accordingly, based on the energetically favorable staggered conformer with possible intramolecular hydrogen bonding between the benzylic hydroxy (at C-7) and aryloxy groups present in a typical 8,40 -oxyneolignan, large and small coupling constant values between H-7 and H-8 correspond to threo and erythro relative configurations, respectively (Gan et al., 2008; Braga et al., 1984; Huang et al., 2013; Huo et al., 2008; Wallis, 1973). The main difference between 1 and the typical 8,40 -oxyneolignans is that the C-7 hydroxy group has now been replaced with a 2hydroxyethylamine side-chain. Examination of models suggested that the NH group in 1 is also able to form a similar intramolecular hydrogen bond with the adjacent aryloxy group (Fig. 3). Since a small coupling constant 3J7,8 = 5 Hz was observed in 1, an erythro relative configuration was suggested for C-7 and C-8. Finally, the configuration of C-8 can be deduced by comparing the CD spectra of 1 and 2 with those of 8,40 -oxyneolignans or related compounds with known absolute configurations. The configuration of C-8 was

HOH 2C Ar

Table 2 H and 13C NMR spectroscopic data of hispiloscine (3) and selected 1H NMR data of 4 (Govindachari et al., 1973) and 5 (Zhen et al., 2002). 1

Position

CH 2CH2 OH N H

7

H

proposed to be 8S by the negative Cotton effects observed at 237 and 235 nm in the CD spectra of 1 and 2, respectively (Arnoldi and Merlini, 1985; Gan et al., 2008; Huang et al., 2013; Huo et al., 2008; Xiong et al., 2011). Consequently, an S configuration can be assigned to C-7 since the relative configuration between C-7 and C-8 was determined as erythro. Therefore, the structure of 1 was elucidated as (+)-(7S,8S,70 E)-3,30 ,4,5,50 -pentamethoxy-7(2-hydroxyethylamino)-8,40 -oxyneolign-70 -ene-9,90 -diol. Hispiloscine (3) was a minor alkaloid obtained from the leaves of F. hispida as a light yellowish oil, [a]D +1.0 (c 0.40, CHCl3). Its UV spectrum showed absorption maxima at 260, 286, 313 and 338 nm indicating the presence of a substituted phenanthrene chromophore (Govindachari, 1973). In addition to the Bohlmann bands observed at 2868 and 2874 cm1, the IR spectrum showed the presence of a strong absorption band at 1736 cm1 suggesting the presence of an ester carbonyl function. The ESIMS of 3 had an [M+H]+ ion at m/z 422, and HRESIMS measurements established the molecular formula as C25H27NO5, requiring 13° of unsaturation. The 13C NMR spectroscopic data of 3 (Table 2) indicated the presence of 25 carbon signals consisting of four methyl, four methylene, two aliphatic methine, five aromatic methine, three oxygenated aromatic methine, six quaternary aromatic and one carbonyl carbon atoms, in agreement with the molecular formula. The most downfield carbon resonance at dC 171.0 supported the presence of an ester function as indicated by the IR spectrum. In addition, the carbon resonances at dC 149.1, 149.6 and 157.5 suggested the presence of three oxygenated aromatic carbons. The 1 H NMR spectroscopic data of 3 (Table 2) indicated the presence of a 1,2,4-trisubstituted aromatic ring from the 1H signals observed at d 7.15 (dd, J = 9, 2 Hz), 7.62 (d, J = 9 Hz), and 7.86 (d, J = 2 Hz), while the two aromatic singlets at d 7.14 and 7.88 suggested the

O H

Ar'

Fig. 3. Staggered conformer of 1 with the lowest energy, viewed along the axis of the C-7–C-8 bond.

a b–e f

3a

4

5

dC

dH (J in Hz)

dH (J in Hz)

dH (J in Hz)

1 2 3 4 4a 4b 5 6 7 8 8a 8b 9

125.7 103.6 157.5 104.7 131.0 124.0 103.8 149.6b 149.1b 115.0 124.5c 129.4 53.5

7.84 7.20 – 7.85 – – 7.88 – – 7.16 – – 3.57 4.75

11

54.5

12

21.7

13

30.1

13a 14 14a 14b 3-OMe 6-OMe 7-OMe

7.86 7.22 – 7.90 – – 7.91 – – 7.19 – – 3.62 4.78 2.45 3.53 1.94 2.02 1.69 2.08 2.71 6.71 – – 4.01 4.11 4.06

14-OCOCH3

66.0 73.5 125.9 124.7c 55.4 55.9d 56.0d 21.2

7.62 7.15 – 7.86 – – 7.88 – – 7.14 – – 3.73 4.48 2.54 3.35 1.88 1.99 2.05 2.21 2.56 6.62 – – 3.99 4.09 4.04 2.03

14-OCOCH3

171.0

–

d (9) dd (9, 2) d (2)

s

s

d (14.7) d (14.7) q (8) t (8) m m m m m d (7.4)

s se se s

d (9) dd (9, 2) d (2)

s

s

d (16) d (16)

f f f f f f f

6.66 – – 3.97 4.09 4.04 2.13 –

d (2)

s s s s

d (9) dd (9, 2) d (2)

s

s

d (16) d (16) m m m m m m m br d

s s s

f

–

CDCl3, 600 MHz; assignments based on COSY, HSQC and HMBC. Assignments are interchangeable. Data not reported.

Please cite this article in press as: Yap, V.A., et al. Hispidacine, an unusual 8,40 -oxyneolignan-alkaloid with vasorelaxant activity, and hispiloscine, an antiproliferative phenanthroindolizidine alkaloid, from Ficus hispida Linn. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.032

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V.A. Yap et al. / Phytochemistry xxx (2014) xxx–xxx

CH 3 H3 CO

3

1 14b

4 4b 5

O

9

H3 CO

6

7

H

13

N

8a

AcO

O

14

12 11

H H

H

H H

H

8

MeO

H H

H H N H

H

MeO

H

H

OMe

OCH3 (

= HMBC)

(

= NOE)

Fig. 4. Selected HMBC correlations and NOEs of 3.

presence of a 1,2,4,5-tetrasubstituted aromatic moiety. The four methyl singlets at dH 2.03, 3.99, 4.04, and 4.09, were readily assigned to an acetyl and three aromatic methoxy groups, respectively. In addition to a pair of AB doublets (J = 14.7 Hz) observed at d 3.73 and 4.48, readily attributable to the isolated benzylic aminomethylene group (H-9), the 1H NMR spectroscopic data also indicated the presence of a highly deshielded aliphatic signal at d 6.62 (d, J = 7.4 Hz), which is characteristic of the benzylic methine H-14 when an acetoxy substituent is present at C-14 (Govindachari et al., 1973; Zhen et al., 2002). In addition to the isolated methylene (H-9) and aromatic CH@CH fragment noted previously, the COSY and HSQC data also showed presence of a CH2CH2CH2CHCH(O) fragment corresponding to the N–C-11–C-12–C-13–C-13a–C-14–Ar partial structure in 3, indicating the presence of an indolizidine moiety with oxygenation at C-14. The substitution pattern of the phenanthrene moiety was established by detailed analysis of the NOE data. Reciprocal NOEs between H-14 and the aromatic doublet at d 7.62 confirmed the latter signal to be due to H-1. This in turn allowed the aromatic resonances at d 7.15 (dd, J = 9, 2 Hz) and d 7.86 (d, J = 2 Hz) to be assigned to H-2 and H-4, respectively. Similarly, reciprocal NOEs between H-9 and the aromatic singlet at d 7.14 confirmed the assignment of the latter signal to H-8, and the other aromatic singlet at d 7.88 to H-5. Thus, C-3, C-6 and C-7 were deduced to be the sites of methoxylation, which were confirmed from the NOEs observed between H-2, H-4/3-OMe, H-5/6-OMe, and H-8/7-OMe (Fig. 4). Finally, attachment of the indolizidine moiety to the trimethoxylated-phenanthrene moiety was established by HMBC data (Fig. 4). Two- and three-bond correlations from H-9 to C-8a, C-8b and C-14a, and H-14 to C-14a and C-8b, allowed the linking of C-9 to C-8b and C-14 to C-14a, respectively, while three-bond correlation from H-14 to the ester carbonyl carbon confirmed the attachment of the acetoxy group at C-14. The proposed structure of hispiloscine (3), which is entirely consistent with the HMBC data (Fig. 4), is identical to those of O-methyltylophorinidine acetate (4) and tylophorinine acetate (5), both of which are semi-synthetic alkaloids derived from O-methyltylophorinidine (4a) and tylophorinine (5a) (Govindachari et al., 1973; Mulchandani and Venkatachalam, 1976; Zhen et al., 2002). The 1H NMR spectroscopic data of 3 present a general similarity to those of 4 and 5 (Table 2), except that the chemical shifts of

O 14a

14

H

13a

N 9

O

OR

H

H-1 (d 7.62), H-9 (d 4.48), H-11 (d 3.35), H-13 (d 2.05 and 2.21), and H-13a (d 2.56) in 3 differ substantially from those of 4 and 5, suggesting possible stereochemical variations at C-13a, C-14 and N (orientation of lone pair electrons) in 3. Further confirmation of this view was the observation that the coupling constant observed for the aliphatic doublet of H-14 in 3 was 7.4 Hz, compared to 2 Hz in 4 and 5 (Govindachari et al., 1973). Hispiloscine (3) was therefore deduced to be diastereomerically related to 4 and 5. Indolizidine heterocycles that contain three stereocenters at C13a, C-14 and N can essentially yield four possibilities of the relative configuration between the two methine hydrogens and the N lone pair electrons, i.e., H-14,H-13a/H-13a,N-Lp: trans/trans, cis/ trans, trans/cis or cis/cis (Fig. 5). In addition to 4 and 4a, other naturally occurring phenanthroindolizidine alkaloids bearing oxygenation at C-14 such as 3-demethyl-14a-hydroxyisotylocrebrine (Abe et al., 1995) and 14a-hydoxy-3,6-didemethylisotylocrebrine (Abe et al., 1998), were previously shown to possess the cis/trans relative configuration. On the other hand, 5 and 5a, as well as the naturally occurring tylophoricidine E (Huang et al., 2002), possessed the trans/cis relative configuration. The 3J13a,14 coupling constant reported for phenanthroindolizidine alkaloids with oxygenation at C-14 and possessing either the cis/trans or trans/ cis configuration were shown to be 0–2 Hz. This is entirely consistent with the coupling constants calculated by a vicinal coupling constant calculator that uses the equation of Haasnoot et al. (1980) and based on the dihedral angles obtained from energyminimized models generated by MM2 calculations (ChemBioDraw 3D software): 4, h = 57°, calcd. 3J13a,14 = 1.5 Hz; 5, h = 84°, calcd. 3 J13a,14 = 1.2 Hz (Fig. 6). By invoking the same argument that the calculated 3J13a,14 values corresponded well with the actual coupling constants, the relative configuration of 3 can be deduced accordingly. Since 3 is a diastereomer of 4 and 5, it can either assume the trans/trans or cis/cis relative configuration. Consequently, the energy-minimized models for the trans/trans and cis/ cis systems were constructed indicating dihedral angles (trans/ trans, h = 152°; cis/cis, h = 41°) that corresponded to the calculated 3 J13a,14 values of 7.0 and 3.2 Hz, respectively (Fig. 6). This strongly suggested that the indolizidine heterocycle in 3 assumed the trans/trans relative configuration, since the doublet of H-14 showed a coupling constant of 7.4 Hz. Furthermore, the presence of a trans indolizidine ring junction in 3 was also corroborated by the observation of the diagnostic Bohlmann bands in the IR spectrum. With regards to the configuration at C-13a, it appeared that phenanthroindolizidine alkaloids with the S configuration exhibited a positive optical rotation (the opposite was observed for alkaloids with the R configuration). Furthermore, variation in the substituents and substitution pattern in both the phenanthrene and indolizidine moieties did not seem to significantly alter the optical rotational properties of the alkaloids (Abe et al., 1995; Buckley and Henry, 1983; Lee et al., 2011; Nordlander and Njoroge, 1987; Stærk et al., 2000; Stoye et al., 2013). On this basis, since a positive optical rotation was observed for 3, the C-13a configuration of 3 can be assigned as S. Taken together, the trans/trans

OR

H

OR

H

12

11

3 (tr ans/t r ans)

N

4 , R = Ac 4a, R = H (cis/tr ans)

N

5, R = Ac 5a, R = H (tr ans/cis)

N

(cis/cis)

Fig. 5. Structures of 3–5.

Please cite this article in press as: Yap, V.A., et al. Hispidacine, an unusual 8,40 -oxyneolignan-alkaloid with vasorelaxant activity, and hispiloscine, an antiproliferative phenanthroindolizidine alkaloid, from Ficus hispida Linn. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.032

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V.A. Yap et al. / Phytochemistry xxx (2014) xxx–xxx

57°

84°

H

H

CH 2 13a

Ar

3J

13a

N

N

cis/trans 1.5 Hz / found 0-2 Hz

3J

13a

Ar

trans /cis 1.2 Hz / found 0-2 Hz

13a,14: calcd.

H3 COCO 152° H CH 2

3

CH2

H 3COCO

OCOCH 3

13a,14: calcd.

Ar

H

H

41°

H

H

H

CH2 13a

Ar

N

trans/trans J13a,14 : calcd. 7.0 Hz / found 7.4 Hz

3

OCOCH3

N

cis/cis J13a,14 : calcd. 3.2 Hz

Fig. 6. Energy-minimized structures of the four possible relative configuration of a C-14-oxygenated phenanthroindolizidine alkaloid, viewed along the axis of the C-13a–C-14 bond and showing the dihedral angles between H-13a and H-14.

at concentrations above 1 lM (data not shown). The mechanism of 1 as a vasorelaxant is not known but other studies of lignans reported selective inhibition of phosphodiesterases. Inhibition of phosphodiesterase V specifically elevated the intracellular level of cGMP which resulted in the vasorelaxant effect observed in the rat aortic rings (Ukita et al., 1999). Hispidacine (1) and hispiloscine (3) were evaluated in vitro against four human cancer cell lines (breast carcinomas MDAMB-231 and MCF-7, lung carcinoma A549, and colon carcinoma HCT-116) and human lung fibroblast MRC-5 cell line via the neutral red uptake assay for their antiproliferative activity. Vinblastine was used as a positive control. As shown in Table 3, alkaloid 1 showed no significant antiproliferative activity against all cell lines tested, while alkaloid 3 showed appreciable activity against all five cell lines (IC50 < 10 lM) (Boik, 2001). The antiproliferative effect of 3 against the colon cancer HCT-116 cell line is notably 3–10-fold greater compared to the other three cancer cell lines and the non-cancerous cell line, indicating that 3 exhibited some degree of selectivity against HCT-116 cells. 3. Conclusions As far as the alkaloid content is concerned, only phenanthroindolizidine and aminocaprophenone alkaloids were previously reported from three Ficus species, namely F. septica, F. fistulosa and F. hispida. From the present study, hispidacine (1) was isolated from the stem-bark of F. hispida as an 8,40 -oxyneolignan that has unusually incorporated a 2-hydroxyethylamine moiety and therefore represents the third alkaloid class isolated from Ficus. Hispiloscine (3), on the other hand, was obtained from the leaves of F. hispida and represents the first naturally occurring phenanthroindolizidine alkaloid with an acetoxy substitution. Hispidacine (1) induced relaxation in rat isolated aorta, while hispiloscine (3) exhibited appreciable antiproliferative activity against breast, lung and colon cancer cell lines. 4. Experimental

Fig. 7. Vasorelaxation effect of 1 and isoprenaline (a b-adrenoceptor agonist) against rat isolated aorta pre-contracted with phenylephrine.

relative configuration and the S-configured C-13a, indicated the configuration at C-14 as R and N as S (a-oriented lone pair electrons). Thus, hispiloscine (3) represents the first naturally occurring acetoxy-substituted phenanthroindolizidine, which is diastereomeric with both the semi-synthetic acetates of O-methyltylophorinidine and tylophorinine. Hispidacine (1) was evaluated for its vasorelaxant activity and was found to produce a concentration dependent relaxation effect on phenylephrine-induced contraction in the isolated rat aortic rings (Fig. 7) with EC50 of 11 lM, which is 100 fold less potent compared to isoprenaline, a non-selective b-agonist (EC50 = 0.1 lM). Although 1 behaves like a ‘partial agonist’ (Emax = 55.6 ± 9.0%) compared to isoprenaline (Emax = 73.6 ± 7.4%), there was no evidence of desensitization (loss of sustained relaxation), unlike isoprenaline

4.1. General experimental procedures Optical rotations were determined on a JASCO P-1020 automatic digital polarimeter. IR spectra were recorded on a Perkin Elmer Spectrum RX1 FT-IR Spectrophotometer. UV spectra were acquired on a GE Ultrospec 8000 spectrophotometer. CD spectra were obtained on a J-815 Circular Dichroism Spectrometer. 1H and 13C NMR spectra were recorded in CDCl3 using TMS as internal standard on a Bruker 600 MHz spectrometer. ESIMS and HRESIMS were obtained on an Agilent 6530 Q-TOF mass spectrometer. 4.2. Plant material Plant material was collected on July 2012 from Selangor, Malaysia, and was identified by K. T. Yong (Institute of Biological Sciences, University of Malaya, Malaysia). Herbarium voucher

Table 3 Cytotoxicity of hispidacine (1) and hispiloscine (3) against five cell lines. Compound

Hispidacine (1) Hispiloscine (3) Vinblastine

Cell lines (IC50, lM) A549

MCF7

MDA-MB-231

HCT 116

MRC-5

75.64 ± 5.28 1.06 ± 0.03 0.010 ± 0.001

50.04 ± 0.46 1.78 ± 0.01 0.001 ± 0.0001

83.12 ± 3.75 1.15 ± 0.01 0.002 ± 0.001

24.75 ± 1.50 0.30 ± 0.02 0.0005 ± 0.00001

Not determined 2.88 ± 0.28 2.27 ± 0.76

A549: human lung carcinoma; MCF-7: estrogen sensitive human breast adenocarcinoma; MDA-MB-231: estrogen insensitive human breast adenocarcinoma; HCT-116: human colorectal carcinoma; MRC-5: human normal lung fibroblast.

Please cite this article in press as: Yap, V.A., et al. Hispidacine, an unusual 8,40 -oxyneolignan-alkaloid with vasorelaxant activity, and hispiloscine, an antiproliferative phenanthroindolizidine alkaloid, from Ficus hispida Linn. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.10.032

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V.A. Yap et al. / Phytochemistry xxx (2014) xxx–xxx

specimens (UNMC77, herbarium No. 48172) are deposited at the Herbarium of the University of Malaya (KLU). 4.3. Extraction and isolation The stem-bark and leaves (3 kg each) of F. hispida were extracted with EtOH:H2O (3 L  4, 95:5, v/v) at room temperature overnight. The concentrated ethanolic extracts were slowly added into 3% tartaric acid solution with constant stirring. The acidic solutions were then filtered through kieselguhr to remove the non-alkaloidal substances. The acidic filtrates were then basified with concentrated NH3 to about pH 10 and the liberated alkaloids were exhaustively extracted with CHCl3, washed with H2O, dried over anhydrous Na2SO4, and concentrated to furnish the crude alkaloidal mixtures (0.67 g and 1.67 g, respectively). The stem-bark alkaloid mixture was initially fractionated by silica gel CC (CHCl3 with increasing proportions of MeOH) to afford eight combined fractions. Fraction-3 was subjected to centrifugal preparative thin layer chromatography (TLC) (CHCl3, NH3-saturated, followed by CH2Cl2/MeOH, NH3-saturated) to yield (+)-deoxypergularinine (5 mg). Fraction-8 was further purified using centrifugal preparative TLC (CHCl3, NH3-saturated) to yield 1 (33 mg). The leaf alkaloid mixture was initially fractionated by silica gel CC (CHCl3 with increasing proportions of MeOH) to afford six combined fractions. Fraction-4 was subjected to centrifugal preparative TLC (CH2Cl2/ MeOH, NH3-saturated, followed by EtOAc/Hexane, NH3-saturated) to yield 3 (7 mg). 4.4. Characterization data 4.4.1. Hispidacine (1) Light yellowish oil; [a]25 D +20 (c 0.28, CHCl3); CD (CH3CN, 0.058 nm), k (de): 206 (+1.29), 237 (2.58), 273 (+1.55) nm; UV (EtOH) kmax (log e) 228 (3.86), 271 (3.60) nm; IR (dry film) mmax 3385, 1585, 1505 cm1; For 13C NMR and 1H NMR spectroscopic data, see Table 1; ESIMS m/z 494 [M+H]+; HRESIMS m/z 494.23801 (calcd for C25H35NO9 + H, 494.23901). 4.4.2. Triacetate derivative of hispidacine (2) A mixture of hispidacine (1) (5 mg, 0.010 mmol), HOAc (10 mol eq.), pyridine (10 mol eq.), 4-dimethylaminopyridine (cat. amount) in CH2Cl2 (3 mL) was stirred under N2 at room temperature for 20 min. The mixture was then poured into saturated Na2CO3 solution and extracted with CH2Cl2. Removal of the solvent, followed by purification by centrifugal preparative TLC over SiO2 (Et2O–MeOH) afforded 4 mg (65%) of the triacetate derivative 2: light yellowish oil; CD (CH3CN, 0.046 nm), k (de): 204 (+1.32), 235 (5.79), 275 (+3.48) nm; IR (dry film) mmax 3325, 1738, 1585, 1505 cm1; For 13C NMR and 1H NMR spectroscopic data, see Table 1; HRESIMS m/z: 620.26907 [M+H]+ (calcd for C31H41NO12 + H, 620.27070). 4.4.3. Hispiloscine (3) Light yellowish oil; [a]25 D +1 (c 0.40, CHCl3); UV (EtOH) kmax (log e) 260 (3.67), 286 (3.51), 313 (3.06), 338 (2.66) nm; IR (dry film) mmax 2874, 2868, 1736 cm1; For 13C NMR and 1H NMR spectroscopic data, see Table 2; ESIMS m/z 422 [M+H]+; HRESIMS m/z 422.19568 (calcd for C25H27NO5 + H, 422.19675).

The thoracic aorta was immediately excised and transferred into cold Krebs–Ringer bicarbonate solution. The Krebs–Ringer bicarbonate solution was freshly prepared daily following the composition (in mM): NaCl 120, KCl 5.4, MgSO4 7H2O 2.4, KH2PO4 1.2, NaHCO3, 25, Glucose 11.7, CaCl2 1.26. All connective tissues were removed from the aorta and cut into 4 mm rings. In a tissue bath, the aorta rings were suspended on metal wire triangles that were connected to a force transducer (MLTF050/ST, ADInstruments, US) via a long surgical suture thread. The measurement of tension was recorded by a PowerLab data acquisition system (LabChart v7.3.4) that displayed on a computer. Aorta rings were maintained in 10 ml Krebs–Ringer bicarbonate solution at 37 °C and aerated with 95% O2, 5% CO2. These rings were allowed to equilibrate for at least 30-min before the application of 2 g wt tension. The aorta rings were pre-contracted with 1  107 M of phenylephrine to achieve 70% of maximal contraction. Once a stable tone was established, cumulative concentration response curves to 1 (1  109 to 1  104 M) and isoprenaline (1  109 to 3  105 M) were determined. Hispidacine (1) was dissolved in DMSO to make 100 mM stock solutions; final bath concentration of DMSO was