Aspidofractinine and Eburnane Alkaloids from a North Borneo Kopsia

Article pubs.acs.org/jnp

Aspidofractinine and Eburnane Alkaloids from a North Borneo Kopsia. Ring-Contracted, Additional Ring-Fused, and PaucidactineType Aspidofractinine Alkaloids from K. paucif lora Wai-Sum Yap,† Chew-Yan Gan,‡ Kae-Shin Sim,§ Siew-Huah Lim,† Yun-Yee Low,† and Toh-Seok Kam*,† †

Department of Chemistry and §Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia University Malaysia of Computer Science and Engineering, Jalan Alamanda 2, Precint 1, 62000 Putrajaya, Malaysia

‡

S Supporting Information *

ABSTRACT: Eleven new indole alkaloids (1−11) comprising seven aspidofractinine and four eburnane alkaloids, were isolated from the stem-bark extract of Kopsia paucif lora occurring in Malaysian Borneo. The aspidofractinine alkaloids include a ring-contracted, an additional ring-fused, a paucidactine regioisomer, two paucidactine, and one kopsine alkaloid. The structures of several of these alkaloids were also confirmed by X-ray diffraction analyses. The bisindole alkaloids isolated, norpleiomutine and kopsoffinol, showed in vitro growth inhibitory activity against human PC-3, HCT-116, MCF-7, and A549 cells and moderate effects in reversing multidrug-resistance in vincristine-resistant human KB cells.

P

lants of the genus Kopsia (Apocynaceae) are distributed mainly over Southeast Asia, India, China, and Australia, with the majority of the species occurring in Southeast Asia.1 Of these, about 16 are found in Malaysia (Peninsular Malaysia and Malaysian Borneo), and several occur both in the Malayan peninsula as well as in Malaysian Borneo.1,2 Plants of this genus are prolific producers of alkaloids, in particular indole and bisindole alkaloids, and are well known to elaborate alkaloids with structurally intriguing molecular skeletons and useful biological activities.3−5 Kopsia paucif lora is documented to occur in various parts of the Malayan peninsula as well as in the Malaysian Borneo state of Sabah.1,2 We previously reported the alkaloid content of samples of K. paucif lora collected in Sabah, Malaysian Borneo, as well as that of samples collected in Johor, Peninsular Malaysia. The Borneo sample (leaf) gave several interesting alkaloids including the pauciflorines, paucidactines, and lahadinines,6−10 while the Malayan sample gave corynanthean, eburnane, seco-leuconoxine, and andranginine-type alkaloids.11,12 Since only a preliminary small-scale investigation was carried out in the earlier study of the stem-bark alkaloids of the North Borneo K. paucif lora,10 we now report the results of a detailed larger scale study, including the isolation, structure determination, and biological activity of 11 new and 28 known alkaloids.

dihydroindole chromophore absorption maxima at 209, 255, and 291 nm, while the IR spectrum showed bands at 3344, 1729, and 1694 cm−1, due to NH, ester, and lactam carbonyl functionalities, respectively. The ESIMS showed an [M + H]+ peak at m/z 339, which analyzed for C20H22N2O3 + H from 13C NMR and HRESIMS data. The 1H and 13C NMR data of 1 (Tables 1 and 2, respectively) showed signals due to the presence of four aromatic hydrogens of an indole moiety (δH 6.70−7.08), an indolic NH (δH 3.91), an isolated aminomethine corresponding to H-21 (δH 3.82, δC 71.5), an isolated methylene α to a carbonyl group (δH 2.04, 2.51; δC 44.7), a methyl ester (δH 3.71, δC 173.2, 52.3), and a lactam (δC 178.7). The COSY spectrum (Figure 1) showed the presence of NCH2CH2, CH2CH, and CH2CH2 partial structures, in addition to the aforementioned isolated aminomethine and methylene groups. The NCH2CH2 fragment corresponds to N−C-5−C-6, from the observed three-bond correlations from H-5 to C-7 and from H-6 to C-8 in the HMBC spectrum. Similarly the characteristic chemical shifts, as well as the HMBC data (Figure 1), indicated that the CH2CH2 and CHCH2 fragments could be assigned to C-18−C-19 and C-16−C-17, respectively, of an aspidofractinine skeleton. The absence of the three-carbon fragment corresponding to N−C-3−C-14−C-15 suggested a change involving the piperidine ring D. The lactam carbonyl is deduced to be at C-14 from the observed three-bond correlations from H-5 and H-21 to C-14 (δC 178.7) in the HMBC spectrum

■

RESULTS AND DISCUSSION Compound 1 (paucidirinine) was initially obtained as a light yellowish oil and subsequently crystallized from CH2Cl2− MeOH as colorless block crystals, mp 180−182 °C, [α]25D −22 (CHCl3, c 0.1). The UV spectrum showed characteristic © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 3, 2015

A

DOI: 10.1021/acs.jnatprod.5b00992 J. Nat. Prod. XXXX, XXX, XXX−XXX

B

1.71 m (α)

1.86 m (β)

3.82 s

19a

19b

21 22 23 24a 24b

12-OMe 16-OH CO2Me

3.71 s

2.07 m (α)

18b

18a

2.18 ddd (14, 10.4, 3.2) (β) 1.37 m (β)

17b

2.04 m (β)

15a

1.60 dd (14, 10.4) (α)

6.70 d (8)

12 14

17a

7.08 m

11

2.94 t (10) (α)

7.08 m 6.77 t (8)

9 10

16

2.74 m

6b

2.51 d (15) (α)

1.43 dd (13.3, 6)

6a

15b

4.01 dd (11.5, 8)

5b

1

3.20 td (11.5, 6)

H

5a

3

4)

6.3)

6.3)

6.3)

3.74 s

1.96 m (β) 2.60 dd (17.5, 7) (α)

3.63 s 4.96 s

1.77 m (β)

1.58 m (α)

1.96 m (α)

2.23 ddd (10, 10, 1) (β) 1.31 t (11.5) (β)

1.55 m (α)

2.95 t (10)

2.02 m (α)

1.08 t (13) (β)

6.71 d (7.7) 2.90 m

7.08 t (7.7)

3.48 dd (11.5, (α) 3.58 dd (11.5, (β) 1.48 dd (13.1, (α) 2.84 dd (12.2, (β) 7.03 d (7.7) 6.77 t (7.7)

2

Table 1. 1H NMR Data (δ) for 1−11a 3

8.74 s

3.03 s

1.61 m (β)

1.33 m (α)

2.29 m (α)

2.50 dd (15, 3.5) (β) 1.61 m (β)

1.89 d (15) (α)

1.52 m

1.19 m

1.26 m 1.75 m

6.82 d (7.5) 6.59 d (7.5)

3.16 m

3.55 t (10)

3.33 m

3.01 m

2.92 m

4

3.70 s

3.49 s

1.99 m

1.34 m

2.04 m

1.34 m

2.04 m

1.34 m

2.95 t (10)

2.40 d (16.3)

1.99 m

6.73 m

7.06 m

2.57 td (12.6, 7.2) 7.06 m 6.73 m

3.44 dd (10, 4.7) 3.63 td (11.8, 4.9) 1.34 m

7.25 s

5

3.72 s

1.71 dd (12.4, 6) 1.71 dd (12.4, 6) 1.90 m

1.40 m

1.90 m

3.01 dd (11.8, 5) 1.50 m

1.59 m

1.48 dd (14, 4)

6.70 d (8) 1.55 m 1.66 m

7.07 t (8)

7.13 d (8) 6.82 t (8)

4.62 s

2.82 td (12.7, 3) 4.24 dd (13.1, 4.5)

2.83 s

2.35 dt (14.5, 3.4) 1.43 dd (11.8, 3.6) 1.80 dd (11.8, 3.6) 1.28 dd (13.6, 4.1) 1.50 m

2.97 dd (12, 3.5) 1.66 m

1.50 m

1.23 m

7.07 td (7.5, 1.4) 6.70 br d (7.5) 1.36 m 1.81 m

7.13 br d (7.5) 6.79 td (7.5, 1)

4.57 dd (5, 3.2)

(10, 5)

(10,

3.84 s

2.26 td (13, 4.5) 1.42 dd (13, 4.5) 1.61 dd (13, 4.5) 3.66 s

1.70 m

1.76 d (9.5)

1.24 m

1.42 dd (13, 4.5) 1.61 dd (13, 4.5) 3.61 d (9.1)

1.54 m 1.67 m

6.89 t (7.7)

6.84 d (7.7) 7.08 t (7.7)

2.99 s

4.22 dd (13, 4.5)

(12,

7 2.88 td (10, 3)

(12.7,

6 2.79 dd 3.2) 3.06 dd 4.5) 3.10 dd 3.2) 3.20 dd

8

4.81 s

2.38 s

2.77 d (17)

2.63 d (17)

1.14 td (13.6, 4) 2.16 d (13.6)

8.32 dd (6, 2) 1.54 m 1.54 m

7.30 td (6, 2)

7.43 dd (6, 2) 7.30 td (6, 2)

2.89 m

2.58 m

3.32 dd (9, 3)

3.32 dd (9, 3)

2.47 dd (16.7, 3.5) 2.63 d (16.7)

9

4.69 s

2.32 s

2.06 dd (14.5, 4) 2.16 d (14.5)

5.99 br d (4)

1.84 td (14, 3.6) 2.16 t (14)

7.43 d (7.7) 7.08 td (7.7, 1) 7.13 td (7.7, 1) 7.38 d (7.7) 1.36 m 1.51 qt (13, 3)

2.97 m

3.27 dd (11.3, 5) 3.27 dd (11.3, 5) 2.54 m

2.56 m

2.56 m

10

4.47 s

4.25 q (6)

1.32 d (6)

5.02 d (8.2)

6.90 d (8.2)

7.33 br d (8) 1.43 d (13.6) 2.10 qt (13.6, 3.6) 1.07 td (14.1, 4.1) 1.94 d (14.1)

7.19 td (8, 1)

7.47 br d (8) 7.12 td (8, 1)

2.83 td (12.5, 3.2) 3.33 dd (8.6, 2.7) 3.33 dd (8.6, 2.7) 2.54 dd (15.4, 1.8) 3.04 m

2.72 m

11

4.18 s 3.70 dq (14, 7) 1.23 t (7)

3.93 q (6)

1.24 d (6)

1.67 dd (14.9, 4) 2.07 d (14.9)

1.67 dd (14.9, 4.5) 1.90 td (14.9, 4.5) 5.51 br d (4)

7.25 d (7) 1.37 br d (14) 2.19 qt (14, 3)

7.17 t (7)

7.46 d (7) 7.12 t (7)

2.98 m

2.60 m

3.29 m

2.64 td (14.9, 5) 3.29 m

2.55 m

Journal of Natural Products Article


Journal of Natural Products

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6

CDCl3, 400 MHz; assignments based on COSY, HSQC, and NOESY. a

5.90 d (1.4) 5.93 d (1.4)

8.94 s

3.91 br s H

NH NCO2Me CHO OCH2O

Table 1. continued

1

3.88 br s

2

3.83 s

3

4

3.89 br s

3.64 s

5

3.54 br s

3.75 s

7

8

9

10

11

(Figure 1), from which it can be inferred that the remaining isolated methylene (C-15) is linked to the lactam carbonyl (C14) and the quaternary C-20. This conclusion is also consistent with the other correlations observed in the HMBC data (3J H21/C-15, H-15/C-19, C-17). Compound 1 is therefore a Dring-contracted kopsinine (or nor-kopsinine) and represents the first example of an aspidofractinine alkaloid, which has lost one carbon in the piperidine ring D. The proposed structure was also confirmed by X-ray diffraction analysis (Figure 2). Compound 2 (paucidirisine) was obtained as a light yellowish oil and subsequently crystallized from CH2Cl2− hexanes as colorless needles, mp 268−270 °C, [α]25D +135 (CHCl3, c 0.1). The UV spectrum showed dihydroindole chromophore absorption maxima at 205, 211, 229, and 279 nm, while the IR spectrum showed bands at 3349 and 1720 cm−1 due to NH and ketone/ester carbonyl functionalities. The ESIMS showed an [M + H]+ peak at m/z 391, and 13C NMR and HRESIMS data established the molecular formula as C24H26N2O3. The 1H and 13C NMR data (Tables 1 and 2, respectively) showed the presence of four aromatic hydrogens of an indole moiety (δH 6.71−7.08), an indolic NH (δH 3.88), an isolated aminomethine corresponding to H-21 (δH 3.63; δC 64.9), an olefinic singlet (δH 4.96), a trisubstituted double bond (δC 96.5, 175.5), a methyl ester (δH 3.74, δC 173.3, 52.2), and a conjugated carbonyl carbon (δC 202.2). The observed downfield shift of the N-substituted tertiary olefinic carbon (C-3) at δC 175.5 and the corresponding upfield shifts of the vinylic carbon and hydrogen (C-22 at δC 96.5, H-22 at δH 4.96) indicated that the double bond is in conjugation with the carbonyl carbon (C-23, δC 202.2). In addition, the observed three-bond correlation from the aminomethine H-21 to the tertiary olefinic C-3 in the HMBC spectrum suggested that the α,β-unsaturated carbonyl function is part of a vinylogous amide unit associated with N-4. The COSY spectrum showed the presence of NCH2CH2, CH2CH2, CHCH2, and CH2CHCH2 partial structures. The NCH2CH2, CH2CH2, and CHCH2 partial structures can be assigned to N−C-5−C-6, C-18−C19, and C-16−C-17, respectively, of an aspidofractinine structure based on their 1H and 13C NMR chemical shifts, as well as the HMBC data (Figure 3). Linking the remaining CH2CHCH2 and NCCH−(CO)− partial structures completed the heptacylic structure of alkaloid 2. The relative configuration at C-14 was deduced from the observed H-14/H21 reciprocal NOEs (Figure 3), which indicated that H-14 has an α-orientation. The proposed structure was also consistent with the full HMBC and NOE data and was also verified by an X-ray diffraction analysis (Figure 4). Compound 2 is notable for the incorporation of an additional cyclopentenone ring fused to the piperidine ring D of an aspidofractinine carbon skeleton. Compound 3 (paucidactinine) was initially obtained as a light yellowish, amorphous solid, which subsequently crystallized from CH2Cl2−hexanes as colorless block crystals, mp 206−208 °C, [α]25D +30 (CHCl3, c 0.2). The IR spectrum showed bands at 3240, 1734, and 1685 cm−1 suggesting the presence of hydroxy, lactone carbonyl, and carbamate functionalities, respectively, while the UV spectrum showed absorption maxima at 210, 224, 250, and 289 nm, indicative of a dihydroindole chromophore. The ESIMS showed an [M + H]+ peak at m/z 441, and 13C NMR and HRESIMS data established the molecular formula as C23H24N2O7. C



Article

Table 2. 13C NMR Data (δ) for 1−11a

a

C

1

2

3

4

5

6

7

8

9

10

11

2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 12-OMe CO2Me CO2Me NCO2Me NCO2Me CHO OCH2O

66.9

66.7 175.5 46.9 34.4 57.1 136.3 121.1 119.8 128.2 111.5 148.9 35.6 38.2 43.9 34.0 33.5 32.2 34.2 64.9 96.5 202.2 41.3

72.3 46.7 54.4 51.2 56.2 132.8 115.1 105.3 149.1 135.7 122.6 15.9 32.5 102.4 49.2 22.3 34.4 33.1 68.3 171.0

66.9 153.0 51.6 35.8 57.3 137.0 121.0 119.5 128.1 111.5 149.3 109.6 29.7 43.6 29.1 33.3 31.6 28.5 64.4

66.1 40.6 167.6 86.0 45.7 132.3 129.2 120.7 121.8 112.1 141.4 20.1 33.9 40.5 27.7 32.0 28.3 32.9 65.1 170.0

66.1 48.0 56.9 89.2 52.5 134.2 122.5 120.1 128.3 111.8 150.1 18.9 34.2 40.3 32.6 29.2 34.1 31.8 68.7 172.9

72.6 40.8 164.9 62.5 56.9 136.1 114.5 126.1 113.5 149.7 131.0 19.6 32.7 51.2 29.2 21.6 32.7 33.3 65.7 204.0

130.9 44.2 50.7 16.6 113.9 130.3 118.4 124.4 124.8 116.4 134.5 22.8 26.9 165.2 42.6 25.8 208.7 52.5 53.9

129.8 44.4 51.0 16.8 105.8 128.7 118.3 120.1 121.3 110.3 135.2 23.1 25.8 73.6 39.3 25.3 211.5 50.0 54.6

128.6 44.9 51.9 16.5 107.0 128.3 118.7 120.3 122.0 108.7 133.7 21.7 26.5 120.1 113.2 18.0 74.4 40.4 58.2

129.4 44.3 51.2 17.0 105.1 128.8 118.5 120.3 121.5 110.8 135.6 21.7 22.7 81.4 35.4 17.8 79.3 37.2 60.1 64.1 15.5

44.8 36.5 55.7 136.3 121.7 120.1 128.2 111.6 149.5 178.7 44.7 43.1 30.6 35.9 28.1 42.3 71.5

56.3 173.2 52.3

173.3 52.2

174.0 52.3 154.9 53.9

152.5 53.0 187.0

100.8

CDCl3, 100 MHz; assignments based on HSQC and HMBC.

Figure 1. COSY, selected HMBCs, and NOEs of 1.

The 1H and 13C NMR data (Tables 1 and 2, respectively) displayed characteristic signals due to two adjacent aromatic hydrogens, corresponding to H-9 and H-10 (δH 6.59, 6.82, J = 7.5 Hz), a methylenedioxy substituent at C-11 and C-12 (δH 5.90, 5.93, J = 1.4 Hz; δC 100.8), a carbamate function (δH 3.83; δC 53.9, 154.9), a lactone carbonyl function (δC 171.0), a secondary carbon linked to two oxygen atoms (δC 102.4), and a hydroxy group at C-16 (δH 8.74). The COSY data (Figure 5) showed the presence of NCH2CH2CH2, NCH2CH, and CH2CH2 partial structures, in addition to the two aromatic hydrogens H-9 and H-10, an isolated aminomethine (NCH, δH 3.03, δC 68.3), and an isolated methylene (δH 1.89, 2.50; δC 49.2). The NMR data (Tables 1 and 2 and Figure 5) indicated an alkaloidal structure of the aspidofractinine group and, in fact, bore a resemblance to those of the paucidactine group of alkaloids, which are characterized by the presence of a lactone function linking C-16 and C-6 [paucidactines A (12), B (13), and C (14)].7,13 The three partial structures shown by the COSY spectrum, therefore, correspond to N−C-3−C-14−C-

Figure 2. X-ray crystal structure of 1.

15, N−C-5−C-6, and C-18−C-19, respectively, while the isolated aminomethine and methylene groups correspond to C21 and C-17, respectively. The additional splitting of H-17β by 3.5 Hz was shown by COSY to be due to W-coupling with H19α. A major difference from the paucidactines, however, was the change in the regiochemistry of the lactone function. In the paucidactines, the lactone carbonyl carbon (C-22) is linked to D



Article

Figure 3. Selected HMBCs and NOEs of 2.


linked by an ester function and with a regiochemistry different from that in the paucidactines. Compound 4 (pauciduridine) was isolated as a light yellowish, amorphous solid, mp 158−160 °C, [α]25D +156 (CHCl3, c 0.7). The UV spectrum showed absorption maxima at 209, 245, and 303 nm, characteristic of a dihydroindole chromophore, while the IR spectrum showed bands at 3349 and 1727 cm−1 suggesting the presence of NH and ester/ formyl carbonyl functionalities, respectively. The ESIMS showed an [M + H]+ peak at m/z 365, which analyzed for C22H24N2O3 + H. The 13C NMR data (Table 2) showed the presence of 22 carbon resonances comprising one methyl, six methylene, and eight methine carbons, two tertiary carbons linked to the indolic nitrogen, one carbonyl carbon, and four quaternary carbon atoms. The methine resonance at δC 187.0 is due to a formyl group, while the carbonyl resonance at δC 174.0 is due to an ester function. The olefinic resonances at δC 153.0 (methine) and 109.6 (quaternary) are due to a trisubstituted double bond. The 1H NMR data (Table 1) showed the presence of four aromatic hydrogens (δH 6.73−7.06), an indolic NH (δH 3.89), a methyl ester (δH 3.70), an isolated aminomethine (δH 3.49; δC 64.4), a vinylic hydrogen (δH 7.25), and a formyl hydrogen (δH 8.94). The observed downfield shift of the formyl resonances (δH 8.94, δC 187.0) suggested conjugation with the trisubstituted double bond, which was also consistent with the deshielding of the β-carbon (C-3) at δC 153.0 versus the α-carbon (C-14) at δC 109.6. The α,β-unsaturated carbonyl unit in fact constitutes part of a vinylogous amide moiety, as shown by the observed three-bond correlations from the vinylic H-3 to C-5, C-21, and CHO, in the HMBC spectrum (Figure 7). The COSY spectrum showed the usual aspidofractinine partial structures such as NCH2CH2, CH2CH2, and CHCH2, in addition to the aminomethine (C21) and an isolated methylene (δH 1.99, 2.40; δC 29.7). Attachment of this methylene to the quaternary olefinic C-14 completes the assembly of the molecule as shown in structure 4, a kopsinine derivative, which has incorporated an additional carbon in the form of a formyl group, constituting part of a vinylogous amide moiety. Compound 5 (paucidactine D) was isolated as a light yellowish oil, [α]25D +208 (CHCl3, c 0.1). The UV spectrum


Figure 5. COSY, selected HMBCs, and NOEs of 3.

C-16, while the ester oxygen is linked to C-6. In alkaloid 3, the lactone carbonyl (C-22) is linked to C-6, while the ester oxygen is linked to C-16. Another difference was the absence of a lactam carbonyl at C-5 in the present compound 3. These differences are consistent with the observed 13C NMR shifts of C-16 and C-6 at δC 102.4 and 51.2, respectively, in 3 (compared to the corresponding shift of C-16 and C-6 in the paucidactines at ca. δC 74 and 83, respectively). This assignment was also supported by the observed three-bond correlation from H-5 to C-22 in 3 and the observed downfield shift of C-16 at δC 102.4, consistent with its attachment to two oxygen atoms. Since suitable crystals were obtained from CH2Cl2−hexanes, X-ray diffraction analysis was carried out (Figure 6), which confirmed the structure based on analysis of the spectroscopic data. Compound 3 represents the first example of an aspidofractinine alkaloid where C-6 and C-16 are E



Article

presence of NCH2CH2CH2, CHCH2, and CH2CH2 partial structures, which were shown by the HMBC data to correspond to N−C-3−C-14−C-15, C-16−C-17, and C-18− C-19, respectively, of an aspidofractinine-type alkaloid. The lactam carbonyl must therefore be at C-5 (δC 167.6), with C-6 being an isolated methine, linked also to an oxygen atom to account for its downfield resonances (δH 4.62; δC 86.0). The remaining oxygen atom must be part of a lactone moiety, with the ester carbonyl branching from C-16. These assignments are supported by the HMBC data (H-6/C-8, C-22; H-17/C-22). Compound 5 (paucidactine D) is therefore a new congener of the paucidactine group of alkaloids. It differs from paucidactine B (13)7 by the absence of the indole methylenedioxy and carbamate substituents. Compound 6 (paucidactine E) was isolated as a light yellowish oil, [α]25D +123 (CHCl3, c 0.2). The UV spectrum showed characteristic dihydroindole absorptions at 210, 242, 257, and 293 nm, while the IR spectrum showed bands at 3336 and 1736 cm−1, corresponding to the presence of NH and lactone carbonyl functionalities, respectively. The ESIMS showed an [M + H]+ peak at m/z 323, which analyzed for C20H22N2O2 + H. The 1H and 13C NMR spectra showed some similarity with those of compound 5, except for the absence of the C-5 lactam carbonyl and the appearance of an NCH2CH fragment, which was shown by COSY and HMBC to correspond to N−C-5−C-6. The characteristic presence of a lactone function with the ester carbonyl linked to C-16 (δH 2.97; δC 40.3), and the ester oxygen to C-6 (δH 4.57; δC 89.2),

Figure 7. Selected HMBCs and NOEs of 4.

showed characteristic dihydroindole absorptions at 209, 243, 257, and 295 nm, while the IR spectrum showed bands at 3332, 1756, and 1698 cm−1, suggesting the presence of NH, lactone, and lactam functionalities, respectively. The ESIMS showed an [M + H]+ peak at m/z 337, which analyzed for C20H20N2O3 + H. The 13C NMR data (Table 2) showed the presence of 20 carbon resonances, comprising six methylenes, seven methines, two tertiary carbons linked to the indolic nitrogen, two carbonyls, and three quaternary carbon atoms. The 13C NMR resonances at δC 170.0 and 167.6 can be assigned to lactone and lactam carbonyl functions, respectively, while the methine resonance at δC 86.0 indicated oxygen substitution at this carbon. The 1H NMR data (Table 1) showed the presence of four aromatic hydrogens (δH 6.70−7.13), an indolic NH (δH 3.64), and an isolated aminomethine corresponding to H-21 (δH 3.72; δC 65.1). The COSY and HMBC data showed the Chart 1

F



Article

m/z 311, and HRESIMS measurements gave the molecular formula C19H22N2O2. The 13C NMR data (Table 2) of 8 showed the presence of 19 carbon resonances comprising one methyl, six methylenes, six methines, two tertiary carbons linked to the indolic nitrogen, one carbonyl, and three quaternary carbon atoms. The NMR data indicated an alkaloid of the eburnane group with OH substitution at C-16 and the presence of an acetyl side chain in place of an ethyl group at C-20. The hydroxymethine H-16 was observed as a broad doublet with J = 4 Hz, indicating that compound 8 belongs to the iso- or epi-eburnamine series of the eburnamine alkaloids.3,15 The NMR data are in fact somewhat similar to those of (+)-isoeburnamine (19) except for the replacement of resonances due to the C-20 ethyl side chain in 19 (δH 0.93; 1.46, 2.19; δC 7.6, 29.0) by those of an acetyl group (δH 2.32, δC 25.3, 211.5) in 8. Compound 8 is therefore readily identified as (−)-19-oxoisoeburnamine. Since the other eburnane alkaloids found in this plant [e.g., (+)-eburnamonine (16), (+)-eburnamenine (17), (−)-eburnamine (18), (+)-isoeburnamine (19), (+)-19-oxoeburnamine (20)] possess the 20β, 21β orientations (or 20R, 21R configuration), it follows that compound 8 possesses the same 20β, 21β orientations, based on a presumed common biosynthetic origin.3,10,15,16 Compound 9 was initially isolated as a light yellowish oil and subsequently crystallized from CH2Cl2−MeOH as light orange block crystals, mp 162−164 °C, [α]25D −157 (CHCl3, c 0.1). The UV spectrum showed indole absorptions at 225, 258, 302, and 309 nm, while the IR spectrum indicated the presence of OH (3364 cm−1). The ESIMS showed an [M + H]+ peak at m/ z 295, and HRESIMS measurements gave the molecular formula C19H22N2O. The 1H and 13C NMR data indicated an eburnane alkaloid showing the presence of 19 carbon resonances, four aromatic hydrogens of an indole moiety (δH 7.12−7.47), an isolated aminomethine corresponding to H-21 (δH 4.47), a cis-disubstituted double bond (H-16, H-17), which is part of an enamine moiety (δH 5.02, 6.90; J = 8.2 Hz; δC 120.1, 113.2), and a hydroxyethyl group (δH 1.32, 3H, d, J = 6 Hz; 4.25, 1H, q, J = 6 Hz). Comparison of the 1H and 13C NMR data showed a similarity to those of (+)-eburnamenine (17),17 except for replacement of the signals of the C-20 ethyl side chain by signals due to a hydroxyethyl group. In common with the other eburnane alkaloids present, the configurations at C-20 and C-21 are assumed to be similar based on a common biosynthetic origin. X-ray diffraction analysis (Figure 9) confirmed the structure deduced, based on the spectroscopic

remained intact as shown by the chemical shift and HMBC data (Figure 8). In addition, the MS data indicated that compound 6

Figure 8. Selected HMBCs of 6.

differed from compound 5 by loss of an oxygen atom and addition of two hydrogens. Compound 6 (paucidactine E) is therefore another new paucidactine-type alkaloid, differing from paucidactine D (5) by the absence of the C-5 lactam carbonyl. Compound 7 (paucidisine) was isolated as a light yellowish oil, [α]25D +88 (CHCl3, c 0.1). The UV spectrum showed characteristic dihydroindole absorptions at 212, 248, and 282 nm, while the IR spectrum showed bands at 1756 and 1693 cm−1, indicating the presence of carbonyl and carbamate/ lactam functionalities, respectively. The ESIMS showed an [M + H]+ peak at m/z 409, which analyzed for C23H24N2O5 + H. The 13C NMR data (Table 2) showed the presence of 23 carbon resonances comprising two methyls, six methylenes, six methines, two tertiary carbons linked to the indolic nitrogen, one tertiary carbon linked to oxygen, three carbonyls, and three quaternary carbon atoms. The 13C NMR resonances at δC 204.0 and 164.9 can be assigned to ketocarbonyl and lactam carbonyl functions, respectively, while the resonance at δC 152.5 can be attributed to the carbonyl of a carbamate functionality. The 1H NMR data showed the presence of three contiguous aromatic hydrogens with methoxy substitution at C12, as shown by the NMR chemical shift and coupling data (Table 1) as well as the HMBC data (3J H-9/C-7, C-11, C-13). Other partial structures shown by COSY include NCH2CH2CH2, CHCH2, and CH2CH2, corresponding to N−C-3−C-14−C-15, C-16−C-17, and C-18−C-19, respectively, of an aspidofractinine skeleton. The aminomethine corresponding to H-21 resonated at δH 3.66 (δC 65.7), while another isolated methine was observed as a singlet at δH 2.99. The N−C-5−C-6 fragment was absent since C-5 is a lactam carbonyl (δC 164.9). The attribution of the singlet at δH 2.99 to H-6 and the observed three-bond HMBC correlations from this hydrogen (H-6) to C-8 and C-21 and from H-21 to C-5 are consistent with this assignment. The remaining ketocarbonyl function is therefore linked from C-6 to C-16, revealing a kopsine-type alkaloid. Compound 7 (paucidisine) is therefore the 12-methoxy-N(1)-carbomethoxy derivative of 5,22-dioxokopsane (15).14 Four new eburnane alkaloids (8−11) were also isolated in addition to the known eburnane alkaloids such as (+)-eburnamonine (16), (+)-eburnamenine (17), (−)-eburnamine (18), (+)-isoeburnamine (19), (+)-19-oxoeburnamine (20), (−)-19(R)-hydroxyisoeburnamine (21), and (+)-19(R)-hydroxyeburnamine (22). Compound 8 was isolated as a light yellowish, amorphous solid, mp 178−180 °C, [α]25D −17 (MeOH, c 0.8). The UV spectrum showed indole absorptions at 209, 227, and 277 nm, while the IR spectrum showed bands at 3315 and 1704 cm−1, suggesting the presence of OH and ketocarbonyl functionalities, respectively. The ESIMS showed an [M + H]+ peak at

Figure 9. X-ray crystal structure of 9. G



Article

of compound 10 as (−)-19(R)-hydroxy-O-ethylisoeburnamine has therefore also provided additional verification of the earlier assignment of the structure of (−)-19(R)-hydroxyisoeburnamine (21).16 We recently reported the structures of the new indole alkaloids larutienine A (23) and its pseudoindoxyl, larutienine B (11), from the Malayan sample of K. paucif lora.11 During the course of the present study, the pseudoindoxyl alkaloid larutienine B (11) was also independently isolated, although in this instance the precursor alkaloid, larutienine A (23), was not detected. In the previous report, the structure of compound 11 was determined based on analysis of the spectroscopic data. In the present study, we obtained suitable crystals of 11 and carried out X-ray diffraction analysis (Figure 11), which confirmed the structure deduced earlier based on analysis of the spectroscopic data.

data, in addition to establishing the configuration at the hydroxy-substituted C-19. Compound 9 is therefore (−)-19(R)-hydroxyeburnamenine. Compound 10 was isolated as a light yellowish oil and subsequently crystallized from CH2Cl2−hexanes as colorless block crystals, mp 150−152 °C, [α]25D −47 (CHCl3, c 0.2). The UV spectrum showed indole absorptions at 226 and 280 nm, while the IR spectrum indicated the presence of OH at 3395 cm−1. The ESIMS showed an [M + H]+ peak at m/z 341, and HRESIMS measurements gave the molecular formula C21H28N2O2. The 1H and 13C NMR data indicated another eburnane alkaloid showing the presence of 21 carbon resonances, four aromatic hydrogens of an indole moiety (δH 7.12−7.46), an isolated aminomethine corresponding to H-21 (δH 4.18), a downfield signal characteristic of the H-16 oxymethine in eburnane alkaloids (δH 5.51, δC 81.4), a hydroxyethyl group (δH 1.24, 3H, d, J = 6 Hz; 3.93, 2H, q, J = 6 Hz), and signals due to an ethoxy group (δH 1.23, 3H, t, J = 7 Hz; 3.70, 2H, dq, J = 14, 7 Hz). The NMR data indicated an eburnane derivative, with a C-20 hydroxyethyl side chain, and a C-16 ethoxy substituent in place of OH. The H-16 resonance was observed at δH 5.51 as a broad doublet with J = 4 Hz, indicating that compound 10 belongs to the iso- or epieburnamine series. Comparison of the NMR data showed a similarity with those of (−)-19(R)-hydroxyisoeburnamine (21), previously isolated by us from another Kopsia species,16 except for the presence of an ethoxy group at C-16 in place of OH. The presence of the ethoxy group strongly suggests that compound 10, (−)-19(R)-hydroxy-O-ethylisoeburnamine, is in all probability an artifact derived from the original alkaloid, (−)-19(R)-hydroxyisoeburnamine (21), since ethanol was used in the extraction of alkaloids. In the previous report, both (−)-19(R)-hydroxyisoeburnamine (21) and (+)-19(R)-hydroxyeburnamine (22) were isolated, but X-ray diffraction was carried out only for the latter since suitable crystals could not be obtained for the former.16 The configuration of C-19 in (−)-19(R)-hydroxyisoeburnamine (21) was at the time assigned based on analogy of the H-19 and C-19 chemical shifts in both alkaloids. In the present instance, suitable crystals of 10 were available, which allowed confirmation of the structure as well as establishment of the C-19 configuration by X-ray diffraction analysis (Figure 10). The present assignment


Three bisindole alkaloids, (−)-norpleiomutine (24), (+)-kopsoffinol (25), and (−)-demethylnorpleiomutine (26), were also isolated in this study, in addition to the monomeric indole alkaloids. These three bisindole alkaloids were also previously isolated by us from another North Borneo Kopsia, K. dasyrachis.16 The same alkaloids in addition to (+)-kopsoffine (27, a diastereomer of 24 with the eburnane half purportedly having the enantiomeric 20S, 21S absolute configuration) were also reported from an earlier study of K. paucif lora.18 In the present study, however, (+)-kopsoffine (27) was not detected. Alkaloids 1−11 did not show any cytotoxicity when tested against drug-sensitive as well as vincristine-resistant KB cells (IC50 > 25 μg/mL). The bisindole alkaloids norpleiomutine (24) and kopsoffinol (25) showed in vitro growth inhibitory activity against human PC-3, HCT-116, MCF-7, and A549 cells (Table 3). In addition, norpleiomutine (24) and kopsoffinol (25) showed moderate effects in reversing multidrug-resistance in vincristine-resistant human KB cells (Table 3). Demethylnorpleiomutine (26), on the other hand, was completely ineffective on both counts. It would appear from these results that the presence of the C-16 hydroxycarbonyl group (as in 26), in place of the methoxycarbonyl group (as in 24), resulted in complete abolition of the biological activity.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Mel-Temp melting point apparatus and are uncorrected. Optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were recorded on a PerkinElmer RX1 FT-IR spectrophotometer. UV spectra were obtained on a Shimadzu UV-3101PC spectrophotometer. 1H and 13C NMR spectra were recorded in CDCl3 using TMS as internal standard on JEOL JNM-LA

Figure 10. X-ray crystal structure of 10. H



Article

Table 3. Cytotoxic Effects of Bisindole Alkaloids 24−26a IC50, μg/mL (μM) compound

KB/S

KB/VJ300

KB/VJ300b

PC-3

HCT-116

MCF-7

A549

(−)-norpleiomutine (24) (+)-kopsoffinol (25) (−)-demethylnorpleiomutine (26) vincristine cisplatin verapamil

14.9 (24.2) 18.2 (28.8) >25 0.0027 (0.0033)

23 (37.3) >25 >25 6.2 (7.5)

4.8 (7.8) 8.6 (13.6) >25

7.1 (11.5) 9.7 (15.3) >25

7.6 (12.3) 15.9 (25.1) >25

9.7 (15.7) 14.1 (22.3) >25

20.4 (33.1) >25 >25

1.5 (5.0)

3.2 (10.7)

4.2 (13.9)

4.3 (14.3)

>25

>25

4.7 (10.3)

a

KB/S: vincristine-sensitive KB carcinoma; KB/VJ300: vincristine-resistant KB carcinoma; PC-3: human prostate carcinoma; HCT-116: human colorectal carcinoma; MCF-7: estrogen-sensitive human breast adenocarcinoma; A549: human lung carcinoma. bWith added vincristine (0.1 μg/mL, 0.121 μM), which did not affect the growth of the KB/VJ300 cells. and 2, respectively; ESIMS m/z 441 [M + H]+; HRESIMS m/z 441.1665 (calcd for C23H24N2O7 + H, 441.1656). Pauciduridine (4): light yellowish, amorphous solid; mp 158−160 °C; [α]25D +156 (c 0.7, CHCl3); UV (EtOH) λmax (log ε) 209 (3.75), 245 (3.63), and 303 (4.24) nm; IR (dry film) νmax 3349, 1727 cm−1; 1 H NMR and 13C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 365 [M + H]+; HRESIMS m/z 365.1858 (calcd for C22H24N2O3 + H, 365.1860). Paucidactine D (5): light yellowish oil; [α]25D +208 (c 0.1, CHCl3); UV (EtOH) λmax (log ε) 209 (3.76), 243 (3.35), 257 (3.08), and 295 (2.96) nm; IR (dry film) νmax 3332, 1756, 1698 cm−1; 1H NMR and 13 C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 337 [M + H]+; HRESIMS m/z 337.1552 (calcd for C20H20N2O3 + H, 337.1547). Paucidactine E (6): light yellowish oil; [α]25D +123 (c 0.2, CHCl3); UV (EtOH) λmax (log ε) 210 (3.92), 242 (3.67), 257 (3.41), and 293 (3.27) nm; IR (dry film) νmax 3336, 1736 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 323 [M + H]+; HRESIMS m/z 323.1758 (calcd for C20H22N2O2 + H, 323.1754). Paucidisine (7): light yellowish oil; [α]25D +88 (c 0.1, CHCl3); UV (EtOH) λmax (log ε) 212 (4.00), 248 (3.46), and 282 (2.86) nm; IR (dry film) νmax 1756, 1693 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 409 [M + H]+; HRESIMS m/z 409.1754 (calcd for C23H24N2O5 + H, 409.1758). (−)-19-Oxoisoeburnamine (8): light yellowish, amorphous solid; mp 178−180 °C; [α]25D −17 (c 0.8, MeOH); UV (EtOH) λmax (log ε) 209 (3.91), 227 (3.99), and 277 (3.43) nm; IR (dry film) νmax 3315, 1704 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 311 [M + H]+; HRESIMS m/z 311.1759 (calcd for C19H22N2O2 + H, 311.1754). (−)-19(R)-Hydroxyeburnamenine (9): light yellowish oil and subsequently light orange block crystals (CH2Cl2−MeOH); mp 162−164 °C; [α]25D −157 (c 0.1, CHCl3); UV (EtOH) λmax (log ε) 225 (4.21), 258 (4.29), 302 (3.79), and 309 (3.79) nm; IR (dry film) νmax 3364 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 295 [M + H]+; HRESIMS m/z 295.1806 (calcd for C19H22N2O + H, 295.1805). (−)-19(R)-Hydroxy-O-ethylisoeburnamine (10): light yellowish oil and subsequently colorless block crystals (CH2Cl2−hexanes); mp 150−152 °C; [α]25D −47 (c 0.2, CHCl3); UV (EtOH) λmax (log ε) 226 (4.11) and 280 (3.75) nm; IR (dry film) νmax 3395 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 341 [M + H]+; HRESIMS m/z 341.2220 (calcd for C21H28N2O2 + H, 341.2224). Larutienine B (11): light yellowish oil and subsequently light orange block crystals (CH2Cl2−MeOH); mp 190−192 °C; [α]25D +414 (c 0.3, CHCl3); UV (EtOH) λmax (log ε) 210 (4.25), 234 (4.55), 257 (3.96), 308 (3.36), and 376 (3.40) nm; IR (dry film) νmax 2855, 2799, 1610 cm−1; 1H NMR and 13C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 311 [M + H]+; HRESIMS m/z 311.1756 (calcd for C19H22N2O2 + H, 311.1754). X-ray Crystallographic Analysis of 1−3 and 9−11. X-ray diffraction analysis was carried out on a Bruker SMART APEX II CCD area detector system equipped with a graphite monochromator and a Mo Kα fine-focus sealed tube (λ = 0.71073 Å), at 100 K. The structure

400 and JNM-ECA 400 spectrometers at 400 and 100 MHz, respectively. HRESIMS data were obtained on an Agilent 6530 QTOF mass spectrometer. Plant Material. Plant material was collected near the Forest Research Centre, Sepilok, Sandakan, Malaysia, and identification was confirmed by Dr. K. M. Wong, Institute of Biological Sciences, University of Malaya. Herbarium voucher specimens (SAN138327) are deposited at the Herbarium, Sabah Forest Department, Sandakan, Sabah. Extraction and Isolation. Extraction of alkaloids from the ground stem-bark material was carried out in the usual standard manner by partitioning the concentrated EtOH extract with 3% tartaric acid. The alkaloids were isolated by initial column chromatography (CC) (SiO2, MeOH−CH2Cl2, increasing percentage of MeOH). This was followed by rechromatography of the appropriate partially resolved fractions using CC or centrifugal preparative TLC. Solvent systems used for centrifugal preparative TLC were CHCl3−hexanes (1:8, NH3saturated), CHCl3−hexanes (1:3, NH3-saturated), CHCl3−hexanes (1:2, NH3-saturated), CHCl3−hexanes (1:1, NH3-saturated), CHCl3− MeOH (100:1, NH3-saturated), Et2O−hexanes (1:9, NH3-saturated), Et2O−hexanes (1:8, NH3-saturated), Et2O−hexanes (1:7, NH3saturated), Et2O−hexanes (1:4, NH3-saturated), Et2O−hexanes (1:2, NH3-saturated), Et2O−hexanes (1:1, NH3-saturated), Et2O−hexanes (3:1, NH3-saturated), and EtOAc−hexanes (1:3, NH3-saturated). The yields (mg kg−1) of the alkaloids were as follows: 1 (0.5), 2 (0. 4), 3 (0.4), 4 (1.3), 5 (0.4), 6 (0.4), 7 (0.6), 8 (0.5), 9 (0.7), 10 (0.5), 11 (0.4), 13 (0.8), 16 (2.5), 17 (2.8), 18 (80.3), 19 (77.3), 20 (1.0), 21 (3.2), 22 (9.8), 24 (176.5), 25 (45.2), 26 (10.4), kopsinine (421.8), tetrahydroalstonine (3.0), leuconoxine (0. 6), N(1)-carbomethoxy5,22-dioxokopsane (1.3), kopsanone (0.5), kopsifine (1.7), decarbomethoxykopsifine (2.1), kopsamine (74.7), kopsamine N-oxide (33.3), N(1)-methoxycarbonyl-12-methoxy-Δ16,17-kopsinine (79.9), N(1)-methoxycarbonyl-12-hydroxy-Δ16,17-kopsinine (0.6), kopsinine N-oxide (45.9), N(1)-methoxycarbonyl-11,12-dimethoxykopsinaline (3.1), kopsilongine (0.4), pleiocarpine (0.9), 12-methoxypleiocarpine (2.9), and pleiocarpine N-oxide (0.7). Paucidirinine (1): light yellowish oil and subsequently colorless block crystals (CH2Cl2−MeOH); mp 180−182 °C; [α]25D −22 (c 0.1, CHCl3); UV (EtOH) λmax (log ε) 209 (4.29), 255 (3.72), and 291 (3.38) nm; IR (dry film) νmax 3344, 1729, 1694 cm−1; 1H NMR and 13 C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 339 [M + H]+; HRESIMS m/z 339.1704 (calcd for C20H22N2O3 + H, 339.1703). Paucidirisine (2): light yellowish oil and subsequently colorless needles (CH2Cl2−hexanes); mp 268−270 °C; [α]25D +135 (c 0.1, CHCl3); UV (EtOH) λmax (log ε) 205 (3.19), 211 (3.13), 229 (2.89), and 279 (2.99) nm; IR (dry film) νmax 3349, 1720 cm−1; 1H NMR and 13 C NMR data, see Tables 1 and 2, respectively; ESIMS m/z 391 [M + H]+; HRESIMS m/z 391.2019 (calcd for C24H26N2O3 + H, 391.2016). Paucidactinine (3): light yellowish, amorphous solid and subsequently colorless block crystals (CH2Cl2−hexanes); mp 206− 208 °C; [α]25D +30 (c 0.2, CHCl3); UV (EtOH) λmax (log ε) 210 (4.01), 224 (4.15), 250 (3.69), and 289 (3.00) nm; IR (dry film) νmax 3240, 1734, 1685 cm−1; 1H NMR and 13C NMR data, see Tables 1 I



Article

was solved by direct methods (SHELXS-97) and refined with fullmatrix least-squares on F2 (SHELXL-2014/7). All non-hydrogen atoms were refined anisotropically, and all hydrogen atoms were placed in idealized positions and refined as riding atoms with relative isotropic parameters. Crystallographic data for compounds 1−3 and 9−11 have been been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44 (0)1223-336033, or e-mail: [email protected]. uk). Crystallographic data of 1: colorless block crystals, C20H22N2O3· CH3OH, Mr = 370.44, orthorhombic, space group P212121, a = 6.7581(2) Å, b = 11.4548(3) Å, c = 23.3157(6) Å; V = 1804.93(9) Å3, T = 100 K, Z = 4, Dcalcd = 1.363 g cm−3, crystal size 0.58 × 0.25 × 0.13 mm3, 16 594 reflections measured (3.494° < 2θ < 54.966°), 4134 unique (Rint = 0.0488, Rsigma = 0.0402). The final R1 value is 0.0361 [I > 2σ(I)] and wR2 = 0.0994 (all data). CCDC deposition number: 1431864. Crystallographic data of 2: colorless needles, C24H26N2O3, CH2Cl2, Mr = 475.39, orthorhombic, space group P212121, a = 6.6620(4) Å, b = 9.8110(5) Å, c = 34.4440(18) Å; V = 2251.3(2) Å3, T = 100 K, Z = 4, Dcalcd = 1.403 g cm−3, crystal size 0.42 × 0.04 × 0.02 mm3, 19 611 reflections measured (2.364° < 2θ < 52.768°), 4609 unique (Rint = 0.1662, Rsigma = 0.1463). The final R1 value is 0.0600 [I > 2σ(I)] and wR2 = 0.1664 (all data). The absolute configuration was determined on the basis of a Flack parameter of 0.01(6), refined using 1930 Friedel pairs. CCDC deposition number: 1431865. Crystallographic data of 3: colorless block crystals, C23H24N2O7, Mr = 440.44, monoclinic, space group P21, a = 9.9492(5) Å, b = 8.2037(4) Å, c = 13.0494(7) Å; β = 109.845(3)°, V = 1002(9) Å3, T = 100 K, Z = 2, Dcalcd = 1.460 g cm−3, crystal size 0.58 × 0.15 × 0.06 mm3, 9224 reflections measured (3.318° < 2θ < 54.998°), 4506 unique (Rint = 0.0461, Rsigma = 0.0661). The final R1 value is 0.0535 [I > 2σ(I)] and wR2 = 0.1282 (all data). CCDC deposition number: 1431866. Crystallographic data of 9: light orange block crystals, C19H22N2O, Mr = 294.39, monoclinic, space group P21, a = 8.6391(10) Å, b = 7.9260(10) Å, c = 11.5438(2) Å; β = 98.4100(10)°, V = 781.945(19) Å3, T = 100 K, Z = 2, Dcalcd = 1.250 g cm−3, crystal size 0.34 × 0.16 × 0.15 mm3, 6701 reflections measured (3.566° < 2θ < 54.992°), 3386 unique (Rint = 0.0316, Rsigma = 0.0525). The final R1 value is 0.0419 [I > 2σ(I)] and wR2 = 0.1068 (all data). CCDC deposition number: 1431867. Crystallographic data of 10: colorless block crystals, C21H28N2O2, Mr = 340.45, orthorhombic, space group P212121, a = 8.4666(3) Å, b = 12.1895(4) Å, c = 16.9938(6) Å; V = 1753.82(10) Å3, T = 100 K, Z = 4, Dcalcd = 1.289 g cm−3, crystal size 0.41 × 0.09 × 0.06 mm3, 15 258 reflections measured (4.112° < 2θ < 52.712°), 3584 unique (Rint = 0.1154, Rsigma = 0.1057). The final R1 value is 0.0603 [I > 2σ(I)] and wR2 = 0.1151 (all data). CCDC deposition number: 1431868. Crystallographic data of 11: light orange block crystals, C19H22N2O2, Mr = 310.39, monoclinic, space group P21, a = 14.2796(4) Å, b = 7.9986(2) Å, c = 15.7780(5) Å; β = 116.781(2)°, V = 1608(8) Å3, T = 100 K, Z = 4, Dcalcd = 1.281 g cm−3, crystal size 0.10 × 0.22 × 0.72 mm3, 15 425 reflections measured (2.892° < 2θ < 54.990°), 7346 unique (Rint = 0.0899, Rsigma = 0.1033). The final R1 value is 0.0473 [I > 2σ(I)] and wR2 = 0.1004 (all data). CCDC deposition number: 1431869. Cytotoxicity Assays. Cytotoxicity assays were carried out following the published procedures.19

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X-ray crystallographic data for compound 3 (CIF) X-ray crystallographic data for compound 9 (CIF) X-ray crystallographic data for compound 10 (CIF) X-ray crystallographic data for compound 11 (CIF)

AUTHOR INFORMATION

Corresponding Author

*Tel: 603-79674266. Fax: 603-79674193. E-mail: tskam@um. edu.my. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the University of Malaya (UMRG) and MOHE Malaysia (HIR-F005) for financial support. REFERENCES

(1) Middleton, D. J. Harv. Pap. Bot. 2004, 9, 89−142. (2) Middleton, D. J. In Flora of Peninsular Malaysia, Series II: Seed Plants; Kiew, R., Chung, R. C. K., Saw, L. G., Soepadmo, E., Boyce, P. C., Eds.; Forest Research Institute Malaysia: Kepong, Malaysia, 2011; Vol. 2. (3) Kam, T. S.; Lim, K. H. In The Alkaloids: Chemistry and Biology; Cordell, G. A., Ed.; Academic Press: The Netherlands, 2008; Vol. 66, pp 1−111. (4) Kam, T. S. In Alkaloids: Chemical and Biological Perspectives; Pelletier, S. W., Ed.; Pergamon: Amsterdam, 1999; Vol. 14, pp 285− 435. (5) Kam, T. S.; Choo, Y. M. In Alkaloids; Cordell, G. A., Ed.; Academic Press: Amsterdam, 2006; Vol. 63, pp 181−337. (6) Kam, T. S.; Yoganathan, K.; Koyano, T.; Komiyama, K. Tetrahedron Lett. 1996, 37, 5765−5768. (7) Kam, T. S.; Yoganathan, K.; Chen, W. Tetrahedron Lett. 1996, 37, 3603−3606. (8) Kam, T. S.; Yoganathan, K. Phytochemistry 1997, 46, 785−787. (9) Kam, T. S.; Yoganathan, K.; Chen, W. J. Nat. Prod. 1997, 60, 673−676. (10) Kam, T. S.; Arasu, L.; Yoganathan, K. Phytochemistry 1996, 43, 1385−1387. (11) Gan, C. Y.; Yoganathan, K.; Sim, K. S.; Low, Y. Y.; Lim, S. H.; Kam, T. S. Phytochemistry 2014, 108, 234−242. (12) Low, Y. Y.; Gan, C. Y.; Kam, T. S. J. Nat. Prod. 2014, 77, 1532− 1535. (13) Lim, K. H.; Hiraku, O.; Komiyama, K.; Koyano, T.; Hayashi, M.; Kam, T. S. J. Nat. Prod. 2007, 70, 1302−1307. (14) Feng, X. Z.; Kan, C.; Potier, P.; Kan, S. K.; Lounasmaa, M. Planta Med. 1983, 48, 280−282. (15) Kam, T. S.; Tan, P. S.; Chen, W. Phytochemistry 1993, 33, 921− 924. (16) Kam, T. S.; Subramaniam, G.; Chen, W. Phytochemistry 1999, 51, 159−169. (17) Atta-Ur-Rahman; Zaman, K.; Perveen, S.; Habib-Ur-Rehman; Muzaffar, A.; Choudhary, M. I.; Pervin, A. Phytochemistry 1991, 30, 1285−1293. (18) Kan, F. C.; Sevenet, T.; Husson, H. P.; Chan, K. C. J. Nat. Prod. 1985, 48, 124−127. (19) Lim, S. H.; Low, Y. Y.; Sinniah, S. K.; Yong, K. T.; Sim, K. S.; Kam, T. S. Phytochemistry 2014, 98, 204−215.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00992. 1 H and 13C NMR for compounds 1−11 (PDF) X-ray crystallographic data for compound 1 (CIF) X-ray crystallographic data for compound 2 (CIF) J


Aspidofractinine and Eburnane Alkaloids from a North Borneo Kopsia

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