Î²-Agarofurans and Sesquiterpene Pyridine Alkaloids from Maytenus

Article pubs.acs.org/jnp

β‑Agarofurans and Sesquiterpene Pyridine Alkaloids from Maytenus spinosa Fátima Gutiérrez-Nicolás,†,‡ Juan C. Oberti,‡,§ Á ngel G. Ravelo,† and Ana Estévez-Braun*,† †

Instituto Universitario de Bio-Orgánica Antonio González (CIBICAN), Departamento de Química Orgánica, Universidad de La Laguna, La Laguna 38206, Tenerife, Spain ‡ Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba X5000HUA, Argentina § Instituto Multidisciplinar de Biología Vegetal (IMBIV; CONICET-UNC), Casilla de Correo 495, Córdoba X5000HUA, Argentina S Supporting Information *

ABSTRACT: Nine new β-dihydroagarofurans (1−9) and four new sesquiterpene pyridine alkaloids (10−13) were isolated from the leaves of Maytenus spinosa. Their structures were determined mainly by 1D- and 2D-NMR spectroscopic studies. The absolute configuration of compound 6 was established using CD spectroscopy. Several derivatives (14−20) were prepared from the sesquiterpene 13. Most of the sesquiterpenoids were tested for anti-HIV activity, but only compound 1 was found to be active. he β-dihydroagarofurans constitute a large group of unusually highly oxygenated sesquiterpenoids, based on the 5,11-epoxy-5β,10α-eudesman-4-(14)-ene skeleton. They are well recognized as chemotaxonomic markers or indicators of the family Celastraceae,1 although some β-dihydroagarofurans have recently been isolated from the Santalaceae species Osyris lanceolata.2 They display a broad spectrum of biological activities including insecticidal, 3 MDR-reversing activity,4,5cytotoxicity,6 antitumor-promoting,7 antitubercular,8 antiHIV,9 immunosuppressive,10,11 and anti-inflammatory effects.12 These properties along with structural characteristics such as their chemical diversity and complex stereochemistry have caused β-dihydroagarofurans to be regarded as privileged structures.13 As part of an ongoing research program to isolate bioactive compounds from Celastraceae species, widely used in South American folk medicine,9,14,15 the constituents of the leaves of Maytenus spinosa (Grisebach) Lourteig & O’Donell16 were studied. This species is an endemic shrub in Argentina, and it is known popularly as “abriboca”. A decoction from the fresh aerial parts is used in folk medicine in Argentina for stomach diseases, as well as an antidiarrheal remedy.17A previous phytochemical study of the root bark of M. spinosa revealed the presence of celastroids, celastroid dimers, pentacyclic triterpenoids, and β-dihydroagarofuran alkaloids.18

T

© 2014 American Chemical Society and American Society of Pharmacognosy

In this report, the isolation and structural elucidation of nine new dihydroagarofurans (1−9) and four new sesquiterpene pyridine alkaloids (10−13) are described, along with the identification of nine known terpenoids from the leaves of M. spinosa. Owing to the large amount of compound 13 isolated, several transformations were carried out on the hydroxy group at C-6, yielding seven derivatives (14−20). The structures of the isolated compounds and derivatives were determined by a combination of spectroscopic techniques, and the majority of the sesquiterpenoids obtained were tested for their anti-HIV activity.

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RESULTS AND DISCUSSION Repeated chromatography of an EtOH extract of the leaves of M. spinosa on silica gel and Sephadex LH-20 yielded 13 new compounds (1−13) and nine known terpenes, namely, 1α,2α,6β,9α,15α-pentacetoxy-8α-benzoyloxy-β-dihydroagarofuran,19 wilfordine E,20 evonoline,21,22 euonine,23 9′-deacetoxymekongensine,24 lupeol, lupenone, betulin, and germanicol.25 The known compounds were identified by comparison with published spectroscopic and physical data. Compound 1 gave the molecular formula C30H38O11, as determined by HREIMS. The IR spectrum revealed the Received: April 9, 2014 Published: July 24, 2014 1853

dx.doi.org/10.1021/np500317t | J. Nat. Prod. 2014, 77, 1853−1863

Journal of Natural Products

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Chart 1

presence of ester groups (1749 cm−1). The mass spectrum contained fragmentation ions attributable to the presence of benzoate (m/z 105) and several fragments corresponding to the loss of acetate groups (i.e., m/z 514 [M − AcOH]+). This was confirmed by the 1H NMR data (Table 1), which displayed signals for four acetate groups [δH 2.21 (3H, s), 2.07 (3H, s), 2.04 (3H, s), and 1.42 (3H, s)] and one benzoate group [δH 8.00 (2H, dd, J = 7.3, 1.3 Hz), 7.53 (1H, bt, J = 7.2 Hz), and 7.40 (2H, bt, J = 7.7]. The 13C NMR and DEPT spectra (Table 2) indicated that 1 possesses a skeleton based on 15 carbons, three methyl carbons at δC 25.7 (C-12), 30.0 (C-13), and 17.5 (C-14), three methylene carbons at δC 30.5 (C-3), 34.6 (C-6), and 65.2 (C-15), six methine carbons at δC 71.2 (C-1), 69.2 (C2), 32.8 (C-4), 48.5 (C-7), 69.2 (C-8), and 77.8 (C-9), and three quaternary carbons at δC 88.9 (C-5), 53.0 (C-10), and 82.5 (C-11). All these data and the presence of a methyl doublet at δH 1.15 (d, J = 7.4 Hz) suggested that 1 is a 1,2,8,9,15-pentasubstituted-β-dihydroagarofuran sesquiterpene.26 From the 1H−1H COSY spectrum, the doublets at δH 5.67 (J = 3.3 Hz) and 5.54 (J = 2.9 Hz), the multiplet at δH 2.08, and the double doublet at δH 5.38 (J = 3.3, 7.8 Hz) were assigned to H-1, H-2, H-7, and H-8. The location of the ester

groups in the basic skeleton was determined by the observed HMBC correlations (Figure 1a). The three-bond correlations between the protons at δH 5.67 (H-1), 5.54 (H-2), 5.93 (H-9), and 5.01/4.32 (H-15) and the carbonyls of acetate groups were used to locate the four acetoxy groups at C-1, C-2, C-9, and C15, and, consequently, the remaining benzoate was placed at C8. The α- or β-orientation of the hydrogens H-1, H-2, H-6, H-8, and H-9 was determined by analysis of the coupling constants and also by ROESY experiments (Figure 1b). Thus, the double doublet at δ 5.38 corresponding to H-8 showed a NOE effect with H-15, which indicated an axial position for H-8 on the α face. The α-orientation of the H-9 proton was determined by the value of the coupling constant, J8,9 = 7.8 Hz, and the βorientation of H-1 and H-2 was also established by the value of the coupling constant, J1,2 = 3.3 Hz, and on the basis of biogenetic considerations, since all dihydroagarofurans described to date have the same β-orientation for H-1.27 Thus, the structure of compound 1 was established as 1α,2α,9β,15tetracetoxy-8β-benzoyloxy-β-dihydroagarofuran. Compound 2 was isolated as an amorphous solid with a molecular formula of C33H44O11. Its 1H NMR spectroscopic data (Table 1) included signals for three acetate groups (δH 1854



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Table 1. 1H NMR Spectroscopic Data of Compounds 1−9 1a

position

3a

4a

1 2

5.67 d (3.3) 5.54 d (3.3)

5.48 d (4.4) 5.33 brs

5.52 brs 5.72 d (3.8)

5.50 d (5.2) 5.45 m

3a 3b

2.35 mc 1.77 d (14.5) 2.35 mc 5.99 s

2.52 m 1.97 d (11.2) 2.44 m 6.36 s

2.33 mc 1.79 d (14.0) 2.33 mc 6.01 s

2.49 d (3.4) 5.38 m

2.67 brs 5.87 brs

5.55 1.60 1.42 1.24 1.69

d (5.6) s s brs s

4′

2.36 m 1.76 dd (2.1, 14.5) 2.28 m 2.48 m 2.09 m 2.08 m 5.38 dd (3.3, 7.8) 5.93 brs 1.45 s 1.51 s 1.15 d (7.4) 5.01 d (12.7) 4.32 d (12.7) 8.00 dd (7.3,1.3) 7.40 brt (7.7)

5′

7.53 brt (7.2)

6′

7.40 brt (7.7)

7′

8.00 dd (7.3, 1.3)

4 6a 6b 7 8 9 12 13 14 15a 15b 3′

2″ 3″ 4″ 5″ 6″ 7″ 3‴ 4‴ 5‴ 6‴ 7‴ CH3COO-R CH3COO-R CH3COO-R CH3COO-R a

2a

s s s s

7a

8b

9

5.55 d (3.2) 4.19 m

5.60 d (2.2) 4.22 brs

5.50 d (3.4) 4.17 m

2.35 mc 1.89 d (13.7)

2.34 mc 1.89 d (13.0)

2.33 mc 1.42 m

2.28 m 6.00 s

2.32 mc 1.91 d (14.6) 2.32 mc 6.29 s

2.35 m 6.72 s

2.34 mc 6.89 s

2.33 mc 6.74 s

2.55 d (4.1) 5.54 m

2.55 d (4.4) 5.59 t (4.8)

2.64 d (4.2) 5.89 t (4.6)

5.69 d (7.6) 1.67 s 1.52 s 1.34 d (7.9) 1.86 s

5.34 1.57 1.43 1.27 1.69

5.48 1.58 1.44 1.34 1.75

5.61 1.65 1.49 1.38 1.79

8.01 d (7.8)

8.08 d (7.3)

8.03 d (7.6)

8.05 d (7.3)

2.56 d (3.6) 5.69 dd (4.0; 5.7) 5.62 d (6.1) 1.60 s 1.45 s 1.24 d (7.4) 5.41 d (13.1) 4.89 d (13.1) 8.03 d (7.7)

2.70 d (2.5) 5.80 dd (2.2; 4.9) 5.77 d (3.5) 1.49 s 1.25 s 1.25 d (7.8) 5.46 d (13.6) 4.79 d (13.6) 7.98 d (7.6)

2.43d (3.4) 5.62 dd (3.8; 8.2) 5.58 d (8.2) 1.57 s 1.42 s 1.23 d (8.8) 5.31 d (12.0) 4.92 d (12.0) 8.02 d (7.4)

7.43 brt (8.2) 7.54 brt (8.3) 7.43 brt (8.2) 8.01 d (7.8)

7.49 m

7.45 brt (7.5) 7.57 brt (7.6) 7.45 brt (7.5) 8.03 d (7.6)

7.47 brt (7.7)

7.45 brt (7.6)

7.42 m

7.45 brt (7.5)

7.58 brt (7.2)

7.56 m

7.57 brt (7.3)

7.45 brt (7.6)

7.42 m

7.45 brt (7.5)

8.03 d (7.7)

7.98 d (7.6)

8.02 d (7.4)

mc m t (7.3) d (7.0)

2.17 s 2.11 s 1.42 s

7.49 m 7.49 m 8.08 d (7.3)

8.01 d (7.1) 7.49 m 7.49 m 7.49 m 8.01 d (7.1) 7.89 d (7.6) 7.32 m 7.57 m 7.32 m 7.89 d (7.6) 2.17 s 1.43 s

d (3.6) s s d (6.8) s

5.34 d (3.5) 4.17 dd (2.5, 5.2) 2.28 m 1.88 d (13.8)

6a 5.31 d (3.2) 4.19 m

2.35 1.40 0.75 1.14

2.21 2.07 2.04 1.42

5a

d (5.3) s s d (7.6) s

7.59 brt (7.5) 7.47 brt (7.7) 8.05 d (7.3)

7.96 brd (7.7) 7.36 brt (7.7) 7.59 m 7.36 brt (7.7) 7.96 d (7.7)

8.06 7.50 7.59 7.50 8.06

2.14 2.12 2.07 1.45

s s s s

2.13 s 2.12 s 2.11 s

d (5.0) s s d (7.4) s

d (7.6) m m m d (7.6)

2.15 s 1.53 s

2.35 1.67 0.84 0.94

mc m t (7.4) d (7.0)

2.23 s 2.13 s 1.60 s

7.96 7.42 7.56 7.42 7.96

d (8.1) m m m d (8.1)

2.16 s 2.15 s 2.13 s

2.71 2.11 2.03 1.57

s s s s

Spectra recorded in CDCl3 at 400 MHz. bSpectra recorded in CDCl3 at 500 MHz. cOverlapped signals.

2.17, 2.11, and 1.42), singlets for three tertiary methyl groups at δH 1.60, 1.42, and 1.69 (Me-12, Me-13, and Me-15), a secondary methyl group at δH 1.24 (Me-14), and one benzoate group [δH 8.01 (2H, d, J = 7.8 Hz), 7.54 (1H, bt, J = 8.3 Hz), 7.43 (2H, bt, J = 8.2 Hz)]. The high-field region of the 1H NMR spectrum also showed a triplet methyl at δH 0.75 (J = 7.3 Hz, H-4″), a doublet methyl at δH 1.14 (J = 7.0 Hz, H-5″), and two multiplets at δH 2.35 (H-2″) and 1.40 (H-3″) characteristic of a methylbutyrate moiety. Furthermore, seven methine protons at δH 5.99, 5.55, 5.48, 5.38, 5.33, 2.49, and 2.35 were observed. On the basis of COSY correlations, the signals at δH 2.49, 5.38, and 5.55 were assigned to H-7, H-8, and H-9, respectively, and the signals at δH 5.48 and 5.33 were assigned to H-1 and H-2, while the remaining singlet at δH 5.99 was assigned to H-6. The positions of the ester groups were solved by analysis of the HMBC spectrum, with the three acetoxy groups located at C-2, C-6, and C-8. The position of the

benzoate ester at C-1 was established by the correlation observed between the doublet at δH 5.48 (H-1) and the benzoyloxy carbonyl carbon at δC 164.7, and the remaining isobutyrate moiety was located at C-9 on the basis of the observed correlation between the carbonyl carbon at δC 174.9 and the doublet at δ 5.55 attributable to H-9. A ROESY experiment (Figure 2) showing a NOE effect between Me-12 and H-9 supported a β-orientation for H-9, and the βorientation for H-8 was established by the value of the coupling constant, J8,9 = 5.6 Hz. The value of the coupling constant, J1,2 = 4.4 Hz, indicated a H-1ax−H-2eq disposition. Accordingly, the structure of 2 was determined with a polyhydroxy basic skeleton of 15-deoxyalatol27 as 1α-benzoyloxy-2α,6β,8αtriacetoxy-9α-methyllbutyroyloxy-β-dihydroagarofuran. Compound 3 was isolated as an amorphous solid with the molecular formula C40H42O11. Its 1H and 13C NMR spectra were similar to those of compound 2 (Tables 1 and 2). The 1855



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Table 2. 13C NMR Spectroscopic Data of Compounds 1−9 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 1‴ 2″ 3‴ 4‴ 5‴ 6‴ 7‴ CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R a

1a 71.2 69.2 30.5 32.8 88.9 34.6 48.5 69.2 77.8 53.0 82.5 25.7 30.0 17.5 65.2 165.0 128.8 129.9 128.1 133.2 128.1 129.9

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

21.1 21.0 20.9 20.1 170.3 169.8 169.7 169.0

CH3 CH3 CH3 CH3 C C C C

2a 74.6 75.9 30.8 33.2 90.4 74.5 52.4 69.5 70.5 48.1 81.6 25.7 30.4 16.3 11.4 164.7 129.9 129.4 128.2 133.0 128.2 129.4 174.9 41.3 26.3 23.9 13.3

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

21.1 CH3 20.2 CH3 18.0 CH3 169.7 C 169.7 C 169.5 C

3a 76.0 70.1 31.0 33.1 90.4 74.5 52.7 71.4 74.3 48.1 81.7 24.0 30.4 18.4 14.0 165.7 130.0 129.5 128.1 132.8 128.1 129.5 165.2 129.5 129.3 128.1 132.8 128.1 129.3 164.7 129.5 129.3 128.3 132.9 128.3 129.3 21.1 20.2

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

169.8 C 169.8 C

4a

5a

74.4 69.7 31.0 33.4 90.6 74.8 52.4 71.3 76.2 48.5 81.8 24.1 30.6 18.2 13.5 164.8 129.8 129.5 128.5 133.2 128.5 129.5

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

21.3 21.3 20.9 20.4 169.9 169.9 169.8 169.8


78.6 69.4 32.5 33.5 91.1 75.0 52.4 71.1 74.9 48.6 81.6 24.1 30.6 18.6 14.0 164.9 129.9 129.5 128.5 133.2 128.5 129.5


21.3 CH3 20.9 CH3 20.7 CH3 169.9 C 169.8 C 169.7 C

6a 78.6 69.5 32.5 33.5 91.2 74.8 52.9 71.5 75.1 48.6 81.7 24.2 30.6 18.7 14.3 165.0 111.7 129.5 128.3 133.1 128.3 129.5 165.4 111.7 129.7 128.5 133.1 128.5 129.7

7a

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

21.3 CH3 20.7 CH3

169.9 C 169.7 C

79.9 68.6 32.7 32.9 90.8 74.7 53.2 69.4 73.0 51.3 81.1 24.6 30.3 17.0 61.0 164.7 129.2 129.6 128.5 133.4 128.5 129.6 175.9 40.9 26.4 11.5 16.2

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

21.3 CH3 21.2 CH3 20.6 CH3 170.0 C 169.7 C 169.5 C

8a 79.5 68.4 32.5 32.7 90.5 74.6 53.2 70.8 72.5 51.2 81.7 24.2 29.5 16.8 61.0 166.1 129.9 129.6 128.3 133.1 128.3 129.6 164.6 129.1 129.4 128.1 132.9 128.1 129.4

9a

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

79.4 68.4 32.5 32.6 90.6 74.5 52.8 69.7 72.5 51.3 80.8 24.4 30.1 16.8 60.3 164.6 129.0 129.4 128.5 133.3 128.5 129.4


21.1 CH3 21.0 CH3 20.5 CH3

21.3 21.1 20.8 20.4 170.2 169.8 169.6 169.5


170.6 C 169.7 C 169.5 C

Spectra recorded in CDCl3 at 100 MHz. Data based on DEPT, HSQC, and HMBC experiments.

Figure 2. Selected ROESY correlations for compounds 2−8. Figure 1. Selected HMBC (a) and ROESY (b) correlations for compound 1.

established by the HMBC correlations determined, in a similar manner to 1 and 2. Thus, the acetoxy groups were located at C1 and C-6 and the benzoyloxy groups at C-2, C-8, and C-9. The 1 H NMR signals for H-1 and H-2 were very close to those of 2 in terms of coupling patterns and coupling constants, which

main differences were the number, type, and relative position of ester groups. Compound 3 was assigned with three benzoates and two acetates, and the placement of these groups was 1856



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suggested the same orientation, H-1ax−H-2eq. In the ROESY spectrum, significant cross-peaks were observed between Me-12 and H-8 and H-8 and H-9 (Figure 2). All these data led to a determination of the structure of 3 as 1α,6β-diacetoxy2α,8α,9α-tribenzoyloxy-β-dihydroagarofuran. The NMR spectroscopic data of sesquiterpenes 4−6 were closely related to those of 2 and 3, showing the typical substitution pattern of the previously described compounds. The main differences were the type of esters present and/or their relative position. Compound 4 gave a molecular formula C30H38O11. The 1H and 13C NMR spectra revealed the presence of four acetates and a benzoate (Tables 1 and 2), and the HMBC experiment allowed the placement of the acetate groups at C-2, C-6, C-8, and C-9 and the benzoate group at C-1. The β-orientation for H-1, H-2, H-8, and H-9 was established on the basis of the NOE effects detected in the ROESY spectrum (Figure 2) and also by the value of the coupling constants. Accordingly, compound 4 was characterized as the 15-deoxyalatol derivative 1α-benzoyloxy2α,6β,8α,9α-tetraacetoxy-β-dihydroagarofuran. The HREIMS of compound 5 showed a molecular ion at m/z 532 corresponding to the formula C28H36O10. The IR spectrum showed absorption bands for hydroxy (3500 cm−1) and ester (1733 cm−1) groups. Its 1H NMR spectrum (Table 1) displayed signals for methyls of three acetyl groups at δH 2.13 (3H, s), 2.12 (3H, s), and 2.11 (3H, s) and five aromatic protons attributable to one benzoate. The double doublet at δH 4.17 (1H, dd, J = 2.5, 5.2 Hz) attributable to a proton geminal to a hydroxy group was assigned to H-2, on the basis of the COSY correlations observed. The benzoyl substituent was located at C-9 by the HMBC correlation between the carbonyl at δC 164.9 and the signal of H-9 (δH 5.48, d, J = 5.3 Hz), and, consequently, the remaining acetate groups could be located at C-1, C-6, and C-8. The NOE effect between the signal of H-12 (δH 1.60 s) and H-8 (t, J = 4.8 Hz) was used to determine the β-orientation for H-8, and the values of the coupling constants, J1,2 = 3.5 Hz and J8,9 = 5.3 Hz, supported the β-orientation for H-1, H-2, and H-9. All these data were used to determine the structure of 5 as 1α,6β,8α-triacetoxy-9α-benzoyloxy-2α-hydroxy-β-dihydroagarofuran. The sesquiterpene 6 was isolated as an amorphous solid with a molecular formula of C33H38O10. The 1H and 13C NMR spectroscopic data showed that it is very similar structurally to 5, except that an acetyl group at C-8 in compound 5 was replaced by a benzoyl group in compound 6. Due to the presence of two chromophores (benzoate groups), the absolute configuration of 6 was resolved by the dibenzoate chirality method, an extension of the circular dichroism exciton chirality procedure.28 The dihedral angle (−21.6°) between the two chromophores was calculated from molecular mechanics calculations, using the PC model program.29 The CD spectrum showed a split curve with a negative Cotton effect at 236.2 nm (Δε = −14.8) and a second positive effect at 220.6 nm (Δε = +5.6), corresponding to the interaction between the benzoate groups at C-8 and C-9. Therefore, the absolute configuration of 6 was determined as (1R,2S,4R,5S,6R,7R,8R,9S,10S)-1,6-diacetoxy-8,9-dibenzoyloxy-2-hydroxy-β-dihydroagarofuran. This compound was previously obtained from the acetylation of the sesquiterpene 6β-acetoxy-8α,9α-dibenzoyloxy-1α,2α-dihydroxy-β-dihydroagarofuran isolated from Tripterygium wilfordii,30 but this is the first time that 6 has been reported as a natural product with its absolute configuration established. Compound 7 was isolated as an amorphous solid with the molecular formula C33H44O12. The 1H NMR spectroscopic data

(Table 1) included signals for three acetate groups, a benzoate, and a methylbutyrate moiety. These groups were supported from the mass spectrum, which contained fragmentation ions attributable to the presence of benzoate (m/z 105) and methylbutyrate (m/z 85). The 1H NMR spectrum showed a singlet at δH 6.72 characteristic of the proton H-6 geminal to an ester group, and the presence of resonances at δH 5.41 (d, J = 13.1 Hz) and δH 4.89 (d, J = 13.1 Hz) and δC 61.0 in the NMR spectra suggested an oxygenated function at C-15. As in the previous compounds discussed, the complete assignments of the protonated carbons were made by analysis of the HSQC experiment results. The regiosubstitution was determined on the basis of the HMBC correlations observed between the signal of H-8 (δH 5.69 dd, J = 4.0, 5.7 Hz) and the carbonyl at δC 175.9 ((CH3)2CHCOO−), between the signal of the H-9 (δH 5.62 d, J = 6.1 Hz) and the carbonyl at 164.7 (PhCOO−), and between the signals of H-1, H-6, and H-15 and the carbonyl carbons of the acetates. The NOE effect between Me12 and H-8 and H-9 established a H-8β/H-9β disposition, and the value of the coupling constant, J1,2 = 3.2 Hz, indicated a βorientation for H-1 and H-2. Thus, compound 7 was characterized as the alatol derivative 1α,6β,15-triacetoxy-8αmethylbutyroyloxy-9α-benzoyloxy-2α-hydroxy-β-dihydroagarofuran. Compound 8 was isolated with the molecular formula C35H40O12 by HREIMS. Its 1H and 13C NMR spectroscopic data showed it to be very similar to 7. The main difference was the presence of a benzoate group at C-8 instead of the methylbutyrate ester. Therefore, the structure of 8 was assigned as 1α,6β,15-triacetoxy-8α,9α-dibenzoyloxy-2α-hydroxy-β-dihydroagarofuran. Compound 9 gave the molecular formula C30H38O12. The NMR data revealed the presence of four acetate groups (δH 2.71, 2.11, 2.03, 1.57) and a benzoate group [δH 8.02 (2H, d, J = 7.4 Hz), 7.45 (2H, bt, J = 7.5 Hz), 7.57 (1H, bt, J = 7.3 Hz)]. A detailed examination of the 1H−1H COSY spectrum of 9 showed two spin systems, with one of them corresponding to H-1−H-2−H-3−H-4 [5.50 (d, J = 3.4 Hz, H-1), 4.17 (m, H-2), 2.33 (m, H-3a + H-4), 1.42 (m, H-3b)] and the other to an AYX system consisting of H-7 (δH 2.43 d, J = 3.4 Hz), H-8 (5.62 dd, J = 3.8, 8.2 Hz), and H-9 (5.58 d, J = 8.2 Hz). The regiosubstitution of this compound was determined by an HMBC experiment, showing a three-bond correlation between the carbonyl carbon of each acetate group and the protons at δH 5.50 (H-1), 6.74 (H-6), 5.62 (H-8), and 5.31/4.93 (H-15). Consequently, the benzoate ester could be located at C-9. The orientation of H-1, H-2, H-8, and H-9 was determined by analysis of the coupling constants and also by a ROESY experiment. The β-orientation of H-2 was assigned by the value of the coupling constant (J1,2 = 3.4 Hz) characteristic of a H1ax−H-2eq disposition. The hydrogen H-9 showed a NOE effect with the doublet at δH 5.50 (H-1), supporting a β orientation for H-9, and the value of the coupling constant, J8,9 = 8.2 Hz, indicated a H-8ax−H-9eq disposition. Thus, 9 was assigned as the 2α-hydroxymalkanguniol derivative2 1α,6β,8β,15-tetracetoxy-2α-hydroxy-9α-benzoyloxy-β-dihydroagarofuran. A series of β-dihydroagarofuran sesquiterpene alkaloids (10− 13) were also isolated. All of them were found to be diesterified at C-3 and C-13 and bound in the diacids wilfordic acid (2-(3carboxybutyl)nicotinic acid, compound 10) and isowilfordic acid (4-(3-carboxybutyl)nicotinic acid, compounds 11−13), and their NMR spectra exhibited the characteristic three aromatic protons corresponding to the disubstituted pyridine 1857



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Table 4. 13C NMR Spectroscopic Data of Compounds 10− 13

unit, two coupled methylene groups, one doublet methyl, and one methine proton as a multiplet (Tables 3 and 4).31

position

Table 3. 1H NMR Spectroscopic Data of Compounds 10−13 10a

position 1

6.18 d (2.9)

2

5.76 brs

3

5.42 d (2.5)

6

6.92 s

7 8 9 12 13a

2.17 d (3.0) 6.02 dd (3.0, 9.4) 6.09 d (9.4) 1.45 s 5.64 d (11.7)

13b

3.71 d (11.7)

14 15a

1.45 s 5.15 d (13.2)

15b

4.96 d (13.2)

2′ 4′ 5′ 6′

8.15 d (8.0) 6.66 dd (4.7, 7.6) 8.53 d (4.2)

7′a 7′b 8′a 8′b 9′ 10′

3.97 m 3.28 m 2.54 m

OH OH

4.73 brs

CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R

1.95 1.84 1.80 1.76 1.71 1.45

2.69 m 1.30 d (6.9)

s s s s s s

11b 5.75 d (3.2) 5.19 t (3.0) 5.08 d (3.0) 5.37 d (3.0) 3.20 s

12b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R CH3COO-R

13b

5.76 mc

5.51 d (2.9)

5.17 d (3.2)

5.06 d (2.8)

4.97 d (2.2)

4.86 d (2.4)

5.05 d (2.8)

5.10 d (2.3)

2.56 d (2.2) 4.25 dd (3.1, 9.2) 5.76 mc 1.79 s 5.91 d (12.4)

2.48 d (2.4) 5.42 dd (2.8, 9.5) 5.59 d (9.8) 1.58 s 5.79 d (12.5)

3.81 d (12.4)

3.66 d (12.5)

1.85 s 4.69 s

1.64 s 4.72 d (13.2)

5.59 s 1.64 s 5.93 d (12.4) 3.82 d (12.5) 1.94 s 4.95 d (13.0) 4.42 d (13.0) 9.27 s

9.28 brs

9.11 s

7.29 m

7.31 brs

7.18 d (5.0)

8.71 d (5.1) 3.99 m 3.20 m 2.39 m 1.60 m 2.39 m 1.21d (6.2) 6.70 s 6.19 d (3.4) 2.19 s 2.11 s 2.00 s 1.93 s

8.88 brs

8.57 d (5.0)

3.96 2.64 2.36 1.62 2.36 1.19

3.82 2.55 2.27 1.53 2.27 1.07

4.46 d (13.2)

m m m m m d (5.6)

m m m m m d (5.6)

6.46 s 6.09 brs

6.37 s 5.97 d (2.8)

2.19 2.17 2.07 1.89

2.08 2.06 1.94 1.90 1.76

s s s s

s s s s s

10a 72.7 69.1 75.6 69.8 93.9 74.7 49.9 73.2 74.8 51.9 85.4 18.7 69.7 23.2 60.5 162.9 124.7 138.4 120.5 152.5 34.4 33.1 39.3 18.1 175.0 167.0 20.7 20.7 20.2 20.2 20.1 20.0 169.4 169.4 169.1 169.1 168.8 168.2

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

11a

12a

13a

71.9 68.5 74.5 72.0 94.0 75.8 64.5 197.6 78.6 52.1 86.4 18.8 71.4 23.7 59.9 152.0 123.8 155.6 126.7 153.6 29.5 34.7 37.7 19.2 174.6 167.2 21.0 20.6 20.4 20.1

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

72.6 68.9 74.9 71.8 93.0 76.6 53.7 73.8 77.0 50.7 86.3 18.7 75.5 23.4 60.8 151.9 124.4 155.6 126.7 153.2 29.4 34.7 37.8 14.1 174.8 167.0 21.4 21.0 20.6 19.0

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

72.1 68.5 74.4 71.4 92.7 76.5 51.7 72.8 73.9 50.7 86.0 18.6 71.0 23.3 59.9 151.6 123.9 155.1 126.4 153.1 29.0 34.4 37.5 18.4 174.4 168.3 21.3 20.7 20.6 20.4 20.3

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

169.6 169.2 169.2 168.5

C C C C

170.7 169.7 169.3 168.6

C C C C

169.6 169.6 169.4 169.0 166.8

C C C C C

a

Spectra recorded in CDCl3 at 100 MHz. Data based on DEPT, HSQC, and HMBC experiments.

a

Spectra recorded in C6D6 at 400 MHz. bSpectra recorded in CDCl3 at 400 MHz. cOverlapped signals.

Compound 10 exhibited the molecular formula C38H47NO18, and the HREIMS showed a molecular ion at m/z 805. Its 1H and 13C NMR spectra were very similar to those of euonine,23 a hexaacetylated β-dihydroagarofuran sesquiterpene alkaloid also isolated in the present study. The same regiosubstitution was confirmed on the basis of the HMBC correlations between the carbonyl carbons of the acetates and the protons at δH 6.18 (d, J = 2.9 Hz, H-1), 5.76 (brs, H-2), 6.92 (s, H-6), 6.02 (dd, J = 3.0, 9.4 Hz, H-8), 6.09 (d, J = 9.4 Hz, H-9), and 4.96 (d, J = 13.2 Hz, H-15b) (Figure 3a). Comparative analysis of the NOE effects and the coupling constants indicated a different orientation for H-9. Thus, the NOE effect observed between Me-12 and H-9 supported a H-9β orientation instead of H-9α present in euonine (Figure 3b). These data were used to

Figure 3. Selected HMBC (a) and ROESY (b) correlations for compound 10.

establish the structure of 10 as 1α,2α,6β,8β,9α,15-hexacetoxy4β-hydroxy-3β,13-[2′-(3-carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran. 1858



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carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran. Compound 13 showed the molecular ion peak at m/z 763, corresponding to the molecular formula C36H45NO17, and its spectroscopic data revealed that this compound is the acetyl derivative at C-8 of compound 12. The H-8 signal appeared at δH 5.42 (dd, J = 2.8, 9.5 Hz) instead of at δH 4.25, and NOEs observed in the ROESY spectrum agreed with the same orientations for H-1, H-2, H-8, and H-9 as in compound 12. These data were used to establish the structure for this compound as 1α,2α,8β,9α,15-pentacetoxy-4β,6β-dihydroxy3β,13-[4′-(3-carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran. Owing to the unusual large amount of 13 isolated (1.25 g), several derivatizations were made at the hydroxy group at C-6, as shown in Scheme 1. Thus, compound 14 was obtained quantitatively by acetylation of 13 with Ac2O−Pyr, while compounds 15−19 were prepared by acylation of 13 with several acyl chlorides of different size, lipophilicity, and stereoelectronic properties. Oxidation of the hydroxy group at C-6 of 13 with the Jones reagent32 yielded the cyclohexanone 20. Due to the previously reported anti-HIV data for related sesquiterpene pyridine alkaloids,9,33 the new dihydroagarofurans 1−13 and the derivatives 14−20 together with the known sesquiterpenes, 1α,2α,6β,9α,15α-pentacetoxy, 8α-benzoyloxyβ-dihydroagarofuran, wilfordine E, evonoline, euonine, and 9′deacetoxymekongensine, were tested for in vitro anti-HIV activity. Of the compounds tested, compound 1 showed a 64.4% inhibition after 72 h and a 96% cell viability. The results obtained indicate that the presence of an acetate at C-15, a αhydroxy group at C-2, and an acetyl moiety with the βorientation at C-8 are important for the activity.

Compounds 11−13 were found to be diesterified isowilfordic acid derivatives, and the presence of a 3,4disubstituted pyridine unit instead of 1,2-disubstituted pyridine was evident from the 1H and 13C NMR data.33 Compound 11 was obtained as an amorphous solid, and its mass spectrum showed the molecular ion peak at m/z 719. The IR spectrum showed bands for hydroxy (3363 cm−1) and ester (1752 cm−1) groups. Its 1H NMR spectrum revealed the presence of four acetates [δH 2.19 (3H, s), 2.11 (3H, s), 2.00 (3H, s), and 1.93 (3H, s)], and the signal of H-6 at δH 5.37 (d, J = 3.0 Hz) suggested the occurrence of a hydroxy group at C-6. The shift of the singlet corresponding to H-7 at δH 3.20 indicated the presence of a carbonyl group at C-8, which was confirmed by the quaternary carbon resonance observed at δC 197.6 in the 13 C NMR and DEPT spectra. COSY, ROESY, HSQC, and HMBC correlations allowed the structural assignment of 1α,2α,9α,15-tetracetoxy-4β,6β-dihydroxy-8-oxo,3β,13-[4′-(3carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran for compound 11. Compound 12, with the molecular formula C34H43NO16, showed similar 1H and 13C NMR spectroscopic data to those of compound 11, except that it was concluded that the carbonyl group at C-8 was replaced by a hydroxy group (δH 4.25 (1H, dd, J = 3.1, 9.2 Hz, H-8)). The regiosubstitution was confirmed on the basis of the HMBC correlations (Figure 4a). NOE

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a PerkinElmer 241 automatic polarimeter. UV spectra were collected in absolute EtOH on a JASCO V-560 spectrophotometer. CD spectra were run on a Jasco J-600 spectropolarimeter. IR spectra were obtained using a Bruker IFS28/55 spectrophotometer. 1 H and 13C NMR spectra were recorded in CDCl3 or C6D6 at 400 and 100 MHz, respectively, with TMS as the internal standard. The 2DNMR experiments were conducted on a Bruker WP-400 SY NMR spectrometer at 400 MHz. High- and low-resolution mass spectra were obtained on a VG Autospec spectrometer. Macherey-Nagel polygram Sil G/UV254 and Analtech silica gel GF preparative layer with UV254

Figure 4. Selected HMBC (a) and ROESY (b) correlations for compound 12.

effects between H-1/H-2, H-9/H-12, and H-8/H-6 (Figure 4b) and also the value of the coupling constant, J1,2 = 3.2 Hz, supported the orientations H-1β, H-2β, H-6α, H-8α, and H-9β. These data were used to establish the structure of 12 as 1α,2α,9α,15-tetracetoxy-4β,6β,8β-trihydroxy-3β,13-[4′-(3-

Scheme 1. Preparation of Derivatives 15−20 from Sesquiterpene 13a

a

Reagents and conditions: (a) RCl, toluene, NEt3, DMAP, reflux; (b) Jones reagent, acetone. 1859



Article

(1R,2S,4R,5S,6R,7R,8R,9S,10S)-1,6-Diacetoxy-8,9-dibenzoyloxy-2hydroxy-β-dihydroagarofuran (6): amorphous, pale yellow solid; [α]20D −49.9 (c 0.42, CHCl3); UV (EtOH) λmax (log ε) 273 (2.75), 230 (3.80) nm; CD λmax (MeCN) nm (Δε) 236.2 (−14.8), 220.6 (+56); IR (neat) νmax 3526, 2930, 1728, 1451, 1369, 1316, 1232, 1096, 1028, 972, 862, 756, 712, 609 cm−1; 1H NMR (CDCl3), see Table 1; 13 C NMR (CDCl3), see Table 2; EIMS m/z 594 [M]+ (1), 552 (6), 472 (2), 430 (6), 351 (3), 294 (5), 248 (5), 230 (5), 190 (5), 105 (100), 77 (16); HREIMS m/z 594.2473 (calcd for C33H38O10, 594.2465). 1α,6β,15-Triacetoxy-8α-methylbutyroyloxy-9α-benzoyloxy-2αhydroxy-β-dihydroagarofuran (7): amorphous, pale yellow solid; [α]20D −4.4 (c 0.64, CHCl3); UV (EtOH) λmax (log ε) 274 (2.75), 231 (3.80) nm; IR (neat) νmax 3495, 2934, 1730, 1453, 1370, 1317, 1234, 1096, 1043, 938, 876, 757, 713, 606, 467 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 2; EIMS m/z 632 [M]+ (1), 590 (8), 490 (4), 409 (4), 397 (7), 260 (7), 246 (9), 218 (11), 149 (12), 109 (10), 105 (100), 85 (25), 57 (39); HREIMS m/z 632.2858 (calcd for C33H44O12, 632.2833). 1α,6β,15-Triacetoxy-8α,9α-dibenzoyloxy-2α-hydroxy-β-dihydroagarofuran (8): amorphous, pale yellow solid; [α]20D −15.6 (c 0.34, CHCl3); UV (EtOH) λmax (log ε) 272 (0.21), 229 (3.47) nm; IR (neat) νmax 3498, 2924, 2853, 1731, 1452, 1370, 1314, 1231, 1096, 1042, 967, 871, 755, 712, 605, 469 cm−1; 1H NMR (CDCl3, 500 MHz), see Table 1; 13C NMR (CDCl3), see Table 2; EIMS m/z 652 [M]+ (1), 610 (5), 592, (3), 105 (100); HREIMS m/z 652.2545 (calcd for C35H40O12, 652.2520). 1α,6β,8β,15-Tetracetoxy-9α-benzoyloxy-2α-hydroxy-β-dihydroagarofuran (9): amorphous, pale yellow solid; [α]20D −1.6 (c 0.31, CHCl3); UV (EtOH) λmax (log ε) 274 (2.93), 230 (3.89) nm; IR (neat) νmax 3441, 2925, 2854, 1737, 1644, 1453, 1371, 1317, 1235, 1097, 1045, 967, 874, 756, 713, 604 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 2; EIMS m/z 590 [M]+ (1), 548 (7), 530 (2), 488 (2), 409 (2), 397 (2), 353 (2), 306 (2), 246 (5), 218 (6), 188 (6), 105 (100), 83 (8), 77 (2); HREIMS m/z 590.2390 (calcd for C30H38O12, 590.2363). 1α,2α,6β,8β,9α,15-Hexacetoxy-4β-hydroxy-3β,13-[2′-(3carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran (10): amorphous, pale yellow solid; [α]20D −8.9 (c 0.60, CHCl3); UV (EtOH) λmax (log ε) 269 (3.43), 224 (3.89) nm; IR (neat) νmax 3441, 2925, 1644, 1452, 1370, 1257, 1091, 753, 666, 596 cm−1; 1H NMR (C6D6), see Table 3; 13C NMR (CDCl3), see Table 4; EIMS m/ z 805 [M]+ (35), 775 (5), 763 (7), 750 (11), 748 (17), 747 (37), 746 (50), 702 (24), 688 (100), 674 (21), 660 (5), 644 (7), 602 (3), 572 (16); HREIMS m/z 805.2792 (calcd for C38H47NO18, 805.2792). 1α,2α,9α,15-Tetracetoxy-4β,6β-dihydroxy-8-oxo,3β,13-[4′-(3carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran (11): amorphous, pale yellow solid; [α]20D −12.0 (c 0.02, CHCl3); UV (EtOH) λmax (log ε) 283 (2.98), 237 (3.07) nm; IR (neat) νmax 3363, 1752, 1373, 1247, 1222, 1162, 1109, 1041, 961, 863, 727, 594 cm−1; 1H NMR (CDCl3), see Table 3; 13C NMR (CDCl3), see Table 4; EIMS m/z 719 [M]+ (57), 704 (13), 659 (17), 646 (17), 539 (10), 446 (10), 392 (2), 280 (1), 224 (12), 206 (30), 178 (33), 160 (26), 132 (34), 93 (100), 83 (14); HREIMS m/z 719.2441 (calcd for C34H41NO16, 719.2425). 1α,2α,9α,15-Tetracetoxy-4β,6β,8β-trihydroxy-3β,13-[4′-(3carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran (12): amorphous, pale yellow solid; [α]20D −8.0 (c 0.56, CHCl3); UV (EtOH) λmax (log ε) 265 (3.01), 223 (3.58) nm; IR (neat) νmax 3391, 2929, 1746, 1457, 1371, 1319, 1228, 1066, 1008, 962, 756, 665, 595 cm−1; 1H NMR (CDCl3), see Table 3; 13C NMR (CDCl3), see Table 4; EIMS m/z 721 [M]+ (20), 706 (36), 704 (7), 662(9), 648 (62), 602 (6), 590 (9), 560 (8), 478 (2), 396 (2), 233 (4), 224 (12), 206 (24), 178 (21), 160 (19), 132 (15), 93 (100), 69 (6); HREIMS m/z 721.2555 (calcd for C34H43NO16, 721.2582). 1α,2α,8β,9α,15-Pentacetoxy-4β,6β-dihydroxy-3β,13-[4′-(3carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran (13): amorphous, white solid; [α]20D −4.9 (c 3.30, CHCl3); UV (EtOH) λmax (log ε) 265 (3.19), 225 (3.71) nm; IR (neat) νmax 3391, 2978, 1649, 1598, 1461, 1374, 1285, 1072, 770, 670, 624, 598, 556 cm−1; 1H NMR (CDCl3), see Table 3; 13C NMR (CDCl3), see Table

were used for TLC. Silica gel (0.2−0.63 mm) and Sephadex LH-20 were used for column chromatography. Plant Material. Leaves of Maytenus spinosa (Griseb) Lourteig et O’Donel (Celastraceae) were collected in Osma, Argentina, at 1250− 1300 m.a.s.l. in October 2005. The plant was identified by Prof. Lázaro Novara (Universidad Nacional de Salta, Argentina), and a voucher specimen was deposited at the herbarium (UNS, Salta, Argentina; account number 12281). Extraction and Isolation. The dried leaves of M. spinosa (910 g) were extracted with EtOH in a Soxhlet apparatus and concentrated under reduced pressure. The EtOH extract was partitioned into a CH2Cl2−H2O (1:1, v/v) solution. The organic phase was dried on anhydrous sodium sulfate, and after filtration and elimination of the solvent, 105 g of a crude extract was obtained. This extract was chromatographed on a silica gel column using an n-hexane−EtOAc gradient ranging from 100% n-hexane to 100% EtOAc, and 20 fractions were obtained. These fractions were repeatedly chromatographed on Sephadex LH-20, silica gel, preparative TLC, and a Chromatotron apparatus (Harrinson Research Inc. USA), to afford lupeol (2.87 g), lupenone (97.0 mg), betulin (101.0 mg), germanicol (97.0 mg), 1α,2α,6β,9α,15α-pentacetoxy-8α-benzoyloxy-β-dihydroagarofuran (32.0 mg), wilfordine E (112.5 mg), evonoline (4.4 mg), euonine (11.5 mg), 9′-deacetoxymekongensine (108.8 mg), 1 (118.1 mg), 2 (3.0 mg), 3 (2.0 mg), 4 (13.4 mg), 5 (8.2 mg), 6 (8.5 mg), 7 (12.7 mg), 8 (8.4 mg), 9 (6.2 mg), 10 (2.7 mg), 11 (12.1 mg), 12 (31.9 mg), and 13 (1.25 g). 1α,6α,9β,15-Tetracetoxy-2α-hydroxy-8α-benzoyloxy-β-dihydroagarofuran (1): amorphous, pale yellow solid; [α]20D +25.5 (c 0.41, CHCl3); UV (EtOH) λmax (log ε) 282 (2.72), 231 (3.89) nm; IR (neat) νmax 2937, 1749, 1455, 1373, 1319, 1245, 1097, 1030, 935, 881, 760, 718, 608 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 2; EIMS m/z 574 [M]+ (2), 559 (12), 532 (57), 514 (2), 410 (7), 335 (3), 275 (4), 262 (7), 220 (10), 202 (10), 173 (11), 105 (100), 77 (14); HREIMS m/z 574.2379 (calcd for C30H38O11, 574.2414). 1α-Benzoyloxy-2α,6β,8α-triacetoxy,9α-methylbutyroyloxy-β-dihydroagarofuran (2): amorphous, pale yellow solid; [α]20D −22.7 (c 0.15, CHCl3); UV (EtOH) λmax (log ε) 274 (2.98), 231 (3.99) nm; IR (neat) νmax 2927, 1743, 1453, 1232, 1095, 1028, 971, 854, 714, 607, 473 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 2; HREIMS m/z 616 [M]+ (3), 587 (3), 574 (17), 560 (14), 556 (5), 472 (3), 452 (1), 372 (3), 290 (2), 279 (5), 249 (4), 215 (7), 173 (7), 149 (22), 105 (100), 85 (23), 71 (24); HREIMS m/z 616.2907 (calcd for C33H44O11, 616.2884). 1α,6β-Diacetoxy-2α,8α,9α-tribenzoyloxy-β-dihydroagarofuran (3): amorphous, pale yellow solid; [α]20D −11.0 (c 0.10, CHCl3); UV (EtOH) λmax (log ε) 279 (3.28), 229 (4.46) nm; IR (neat) νmax 2926, 1725, 1450, 1275, 1095, 1027, 969, 711, 471 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 2; EIMS m/z 698 [M]+ (1), 656 (11), 593 (1), 576 (3), 534 (3), 474 (1), 379 (1), 279 (1), 230 (2), 215 (3), 173 (2), 105 (100), 77 (10); HREIMS m/z 698.2758 (calcd for C40H42O11, 698.2727). 1α-Benzoyloxy-2α,6β,8α,9α-tetraacetoxy-β-dihydroagarofuran (4): amorphous, pale yellow solid; [α]20D −36.7 (c 0.67, CHCl3); UV (EtOH) λmax (log ε) 274 (2.77), 231 (3.86) nm; IR (neat) νmax 2927, 1744, 1451, 1369, 1317, 1235, 1096, 1030, 970, 873, 757, 714, 605 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 2; EIMS m/z 574 [M]+ (4), 532 (33), 514 (8), 472 (7), 453 (5), 410 (4), 350 (2), 279 (4), 249 (4), 220 (6), 173 (6), 105 (100), 77 (11); HREIMS m/z 574.2403 (calcd for C30H38O11, 574.2414). 1α,6β,8α-Triacetoxy-9α-benzoyloxy-2α-hydroxy-β-dihydroagarofuran (5): amorphous, pale yellow solid; [α]20D −27.2 (c 0.41, CHCl3); UV (EtOH) λmax (log ε) 274 (3.03), 229 (4.07) nm; IR (neat) νmax 3500, 2931, 1733, 1452, 1369, 1317, 1233, 1096, 1031, 971, 869, 755, 713, 604, 474 cm−1; 1H NMR (CDCl3), see Table 1; 13 C NMR (CDCl3), see Table 2; EIMS m/z 532 [M]+ (2), 490 (10), 472 (4), 430 (9), 351 (5), 220 (6), 195 (6), 153 (6), 133 (8), 105 (100), 77 (14); HREIMS m/z 532.2323 (calcd for C28H36O10, 532.2308). 1860



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4; EIMS m/z 763 [M]+ (6), 748 (12), 704 (7), 690 (28), 632(4), 224 (6), 206 (13), 178 (15), 160 (12), 132 (10), 93 (100), 69 (7); HREIMS m/z 763.2681 (calcd for C36H45NO17, 763.2687). 1α,2α,6β,8β,9α,15-Hexacetoxy-4β-hydroxy-3β,13-[4′-(3carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran (14). To 20.0 mg (0.026 mmol) of compound 13 dissolved in pyridine (1.5 mL) were added 0.3 mL (3.18 mmol) of Ac2O and a catalytic amount of 4-(dimethylamino)pyridine. The reaction mixture was stirred for 24 h at room temperature; then it was carried to dryness under reduced pressure and purified by preparative TLC with a mixture of n-hexane−EtOAc (8:2) to give compound 14 (20.3 mg, 100%) as an amorphous, white solid: [α]20D −15.8 (c 0.1, CHCl3); UV (EtOH) λmax (log ε) 264 (3.35); 225 (3.88) nm; IR (neat) νmax 2925, 1750, 1592, 1553, 1370, 1228, 1164, 1099, 1043, 969, 756, 592, 472 cm−1; 1H NMR (CDCl3, 400 MHz) δ 9.19 (1H, s, H-2′), 8.69 (1H, d, J = 5.0 Hz, H-6′), 7.29 (1H, d, J = 5.0 Hz, H-5′), 6.63 (1H, s, H-6), 5.78 (1H, d, J = 12.0 Hz, H-13a), 5.59 (1H, d, J = 9.8 Hz, H-9), 5.63 (1H, d, J = 2.9 Hz, H-1), 5.62 (1H, m, H-8), 5.18 (1H, t, J = 3.1 Hz, H-2), 5.12 (1H, d, J = 1.2 Hz, OH), 4.93 (1H, d, J = 2.9 Hz, H-3), 4.74 (1H, d, J = 13.0 Hz, H-15a), 4.69 (1H, d, J = 13.0 Hz, H-15b), 3.81 (2H, d, J = 12.0 Hz, H-13b y H-7′a), 2.71 (1H, m, H-7′b), 2.50 (1H, d, J = 3.1 Hz, H-7), 2.40 (1H, m, H-9′), 2.33 (3H, s, CH3COO), 2.21 (1H, m, H-8′a), 2.19 (3H, s, CH3COO), 2.16 (3H, s, CH3COO), 2.05 (3H, s, CH3COO), 2.01 (3H, s, CH3COO), 1.87 (3H, s, CH3COO), 1.73 (3H, s, Me-14), 1.70 (1H, m, H-8′b), 1.58 (3H, s, Me-12), 1.22 (3H, d, J = 7.1 Hz, Me-10′); 13C NMR (CDCl3, 100 MHz) δ 174.4 (C, C-11′), 170.0 (C, CH3COO), 169.8 (C, CH3COO), 169.7 (C, CH3COO), 169.7 (C, CH3COO), 169.3 (C, CH3COO), 168.7 (C, C12′), 166.7 (C, CH3COO), 154.7 (C, C-4′), 153.1 (CH, C-6′), 151.8 (CH, C-2′), 126.1 (CH, C-5′), 124.7 (C, C-3′), 94.0 (C, C-5), 86.0 (C, C-11), 75.5 (CH, C-3), 74.5 (CH, C-6), 74.2 (CH, C-9), 73.2 (CH, C-8), 72.5 (CH, C-1), 69.8 (C, C-4), 70.4 (t, C-13), 68.8 (CH, C-2), 60.5 (CH2, C-15), 51.5 (C, C-10), 50.1 (CH, C-7), 38.1 (CH, C-9′), 34.5 (CH2, C-8′), 29.7 (CH2, C-7′), 23.3 (CH3, Me-12), 21.5 (CH3, CH3COO), 21.2 (CH3, CH3COO), 21.0 (CH3, CH3COO), 20.9 (CH 3, CH3 COO), 20.8 (CH3, CH3COO), 20.6 (CH3 , CH3COO), 18.8 (CH3, Me-14), 18.3 (CH3, Me-10′); EIMS m/z 805 [M]+ (64), 788 (5), 746 (4), 439 (6), 307 (17), 286 (100), 224 (3), 206 (7); HREIMS m/z 805.2849 (calcd for C38H47NO18, 805.2793. 1α,2α,8β,9α,15-Pentacetoxy-6β-p-bromobenzoyloxy-4β-hydroxy-3β,13-[4′-(3-carboxybutyl)]nicotinic acid-dicarbolactone-βdihydroagarofuran (15). To 20.0 mg (0.026 mmol) of compound 13 dissolved in dry toluene (3.0 mL) were added 11.07 μL (3.0 equiv) of dry NEt3 and 11.7 mg (2 equiv) of p-bromobenzoyl chloride together with a catalytic amount of 4-(dimethylamino)pyridine. The reaction mixture was stirred for 24 h under reflux; then the solvent was eliminated under reduced pressure, and the resulting residue was purified by preparative TLC using n-hexane−EtOAc (4:1) to give 7.7 mg of compound 15 (31%) as an amorphous, white solid: [α]20D −4.5 (c 0.1, CHCl3); UV (EtOH) λmax (log ε) 224 (3.83) nm; IR (neat) νmax 3447, 2929, 1753, 1648, 1595, 1464, 1375, 1259, 1228, 1182, 1099, 1055, 1015, 912, 851, 761, 669, 597 cm−1; 1H NMR (CDCl3, 400 MHz) δ 9.17 (1H, s, H-2′), 8.72 (1H, brs, H-6′), 8.14 (2H, d, J = 8.6 Hz, H-4″, H-6″), 7.64 (2H, d, J = 8.6 Hz, H-3″, H-7″), 7.34 (1H, d, J = 3.3 Hz, H-5′), 6.69 (1H, s, H-6), 5.77 (3H, m, H-13a, H-8, H-9), 5.68 (1H, d, J = 3.3 Hz, H-1), 5.33 (1H, s, OH), 5.22 (1H, t, J = 3.0 Hz, H-2), 4.98 (1H, d, J = 2.9 Hz, H-3), 4.80 (1H, d, J = 13.2 Hz, H15a), 4.71 (1H, d, J = 13.2 Hz, H-15b), 3.84 (1H, m, H-7′a), 3.71 (1H, d, J = 11.8 Hz, H-13b), 2.76 (1H, m, H-7′b), 2.67 (1H, d, J = 2.8 Hz, H-7), 2.54 (1H, m, H-9′), 2.39 (3H, s, CH3COO), 2.26 (1H, m, H8′a), 2.16 (3H, s, CH3COO), 2.06 (3H, s, CH3COO), 2.02 (3H, s, CH3COO), 1.89 (3H, s, CH3COO), 1.82 (1H, m, H-8′b), 1.75 (3H, s, Me-14), 1.60 (3H, s, Me-12), 1.25 (3H, m, Me-10′); 13C NMR (CDCl3, 100 MHz) δ 174.5 (C, C-11′), 169.9 (C, CH3COO), 169.7 (C, CH3COO), 169.7 (C, CH3COO), 169.3 (C, CH3COO), 168.7 (C, CH3COO), 166.9 (C, C-12′), 165.1 (C, C-1″), 154.3 (C, C-4″), 153.2 (CH, C-6′), 151.7 (CH, C-2′), 132.3 (CH, C-4″ + C-6″), 131.8 (CH, C-3″ + C-7″), 129.2 (CH, C-5′), 128.0 (C, C-3′), 125.9 (C, C5″), 124.9 (C, C-2″), 93.5 (C, C-5), 86.0 (C, C-11), 75.9 (CH, C-6),

75.6 (CH, C-3), 74.4 (CH, C-9), 72.9 (CH, C-8), 72.4 (CH, C-1), 70.3 (CH2, C-13), 70.0 (C, C-4), 68.9 (CH, C-2), 60.5 (CH2, C-15), 51.7 (C, C-10), 50.1 (CH, C-7), 38.3 (CH, C-9′), 34.5 (CH2, C-8′), 29.7 (CH2, C-7′), 23.4 (CH3, Me-12), 21.3 (CH3, CH3COO), 21.0 (CH3, CH3COO), 20.8 (CH3, CH3COO), 20.7 (CH3, CH3COO), 20.6 (CH3, CH3COO), 18.8 (CH3, Me-14), 18.1 (CH3, Me-10′); EIMS m/z 947 [M]+ (94), 945 (84), 932 (36), 887 (40), 874 (74), 872 (67), 866 (29), 746 (100), 716 (7), 688 (16), 644 (9), 602 (6); HREIMS m/z 947. 2018 (cald for C43H48NO1881Br, 947.2034, 945.2019 (calcd for C43H48NO1879Br, 945.2055). 1α,2α,8β,9α,15-Pentacetoxy-6β-nicotinoyloxy-4β-hydroxy-3β,13[4′-(3-carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran (16). To 20.0 mg (0.026 mmol) of compound 13 dissolved in dry toluene (3.0 mL) were added 11.07 μL (3.0 equiv) of dry NEt3, 9.5 mg (2 equiv) of nicotinoyl chloride, and a catalytic amount of 4(dimethylamino)pyridine. The reaction mixture was stirred for 48 h under reflux conditions; then the solvent was eliminated under reduced pressure, and the resulting residue was purified by preparative TLC using n-hexane−EtOAc (4:1) to yield 7.9 mg (34%) of compound 16 as an amorphous, white solid: [α]20D −8.5 (c 0.4, CHCl3); UV (EtOH) λmax (log ε) 263 (3.63), 222 (4.16) nm; IR (neat) νmax 3439, 2925, 1750, 1641, 1593, 1460, 1370, 1227, 1150, 1093, 1049, 907, 755, 594, 491 cm−1; 1H NMR (CDCl3, 400 MHz) δ 9.37 (1H, s, H-2′), 9.14 (1H, s, H-2″), 8.81 (1H, d, J = 4.5 Hz, H-6″), 8.68 (1H, d, J = 6.9 Hz, H-4″), 8.59 (1H, d, J = 5.7 Hz, H-6′), 7.44 (1H, dd, J = 6.2, 10.5 Hz, H-5″), 7.29 (1H, d, J = 6.9 Hz, H-5′), 6.76 (1H, s, H-6), 5.77 (3H, m, OH, H-13a, H-8), 5.66 (1H, d, J = 4.4 Hz, H-9), 5.37 (1H, s, H-1), 5.19 (1H, t, J = 4.1 Hz, H-2), 4.95 (1H, d, J = 3.8 Hz, H-3), 4.76 (1H, dd, J = 9.6, 17.6 Hz, H-15), 3.67 (1H, m, H7′a), 3.64 (1H, d, J = 11.8 Hz, H-13b), 2.73 (1H, m, H-7′b), 2.66 (1H, s, H-7), 2.49 (1H, m, H-9′), 2.37 (3H, s, CH3COO), 2.26 (1H, m, H8′a), 2.14 (3H, s, CH3COO), 2.04 (3H, s, CH3COO), 2.00 (3H, s, CH3COO), 1.87 (3H, s, CH3COO), 1.58 (3H, s, Me-14), 1.53 (3H, s, Me-12), 1.35 (1H, m, H-8′b), 1.17 (3H, d, J = 8.5 Hz, Me-10′); 13C NMR (CDCl3, 100 MHz) δ 174.3 (C, C-11′), 169.8 (C, CH3COO), 169.6 (C, CH3COO), 169.5 (C, CH3COO), 169.1 (C, CH3COO), 168.5 (C, C-12′), 166.6 (C, CH3COO), 164.2 (C, C-7″), 154.5 (C, C4′), 153.9 (CH, C-6′), 152.9 (CH, C-2″), 151.4 (CH, C-2′), 151.3 (CH, C-6″), 137.7 (CH, C-4″), 126.0 (CH, C-5′), 124.7 (C, C-3′), 124.2 (CH, C-3″), 123.7 (C, C-5″), 93.4 (C, C-5), 85.0 (C, C-11), 75.7 (CH, C-3), 75.3 (CH, C-6), 74.0 (CH, C-9), 72.3 (CH, C-8), 72.2 (CH, C-1), 70.1 (CH2, C-13), 69.8 (C, C-4), 68.6 (CH, C-2), 60.2 (CH2, C-15), 51.4 (C, C-10), 49.8 (CH, C-7), 37.9 (CH, C-9′), 34.9 (CH2, C-8′), 29.5 (CH2, C-7′), 23.2 (CH3, Me-12), 21.1 (CH3, CH3COO), 20.8 (CH3, CH3COO), 20.6 (CH3, CH3COO), 20.5 (CH3, CH3COO), 20.4 (CH3, CH3COO), 18.6 (CH3, Me-14), 18.0 (CH3, Me-10′); EIMS m/z 868 [M]+ (100), 854 (42), 808 (12), 805 (6), 746 (23), 644 (8), 602 (6); HREIMS m/z 868.2873 (calcd for C42H48N2O18, 868.2902). 1α,2α,8β,9α,15-Pentacetoxy-6β-furoyloxy-4β-hydroxy-3β,13-[4′(3-carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran (17). To 20.0 mg (0.026 mmol) of compound 13 dissolved in dry toluene (3.0 mL) were added 11.07 μL (3.0 equiv) of dry NEt3, 5.44 μL (2 equiv) of 2-furoyl chloride, and a catalytic amount of 4(dimethylamino)pyridine. The reaction mixture was stirred for 24 h under reflux; then the solvent was eliminated under reduced pressure, and the resulting residue was purified by preparative TLC using nhexane−EtOAc (4:1) to give 24.2 mg (99%) of compound 17 as an amorphous, white solid: [α]20D −17.1 (c 0.3, CHCl3); UV (EtOH) λmax (log ε) 263 (3.33), 220 (3.81) nm; IR (neat) νmax 3471, 2931, 1755, 1595, 1473, 1375, 1232, 1183, 1120, 1055, 912, 762, 598 cm−1; 1 H NMR (CDCl3, 400 MHz) δ 9.14 (1H, brs, H-2′), 8.68 (1H, brs, H6′), 7.63 (1H, d, J = 0.9 Hz, H-5″), 7.46 (1H, d, J = 4.7 Hz, H-3″), 7.28 (1H, s, H-5′), 6.72 (1H, s, H-6), 6.51 (1H, dd, J = 2.2, 4.6 Hz, H4″), 5.86 (1H, d, J = 15.9 Hz, H-13a), 5.64 (2H, s, H-1 and H-9), 5.63 (1H, s, H-8), 5.17 (1H, t, J = 3.9 Hz, H-2), 5.08 (1H, s, OH), 4.92 (1H, d, J = 3.8 Hz, H-3), 4.72 (2H, s, H-15), 3.75 (1H, d, J = 16.0 Hz, H-13b), 3.59 (1H, m, H-7′a), 2.74 (1H, m, H-7′b), 2.68 (1H, s, H-7), 2.47 (1H, t, J = 9.1 Hz, H-9′), 2.35 (3H, s, CH3COO), 2.24 (1H, m, H-8′a), 2.13 (3H, s, CH3COO), 2.03 (3H, s, CH3COO), 1.99 (3H, s, 1861



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(CDCl3, 400 MHz) δ 9.17 (1H, s, H-2′), 8.70 (1H, d, J = 4.9 Hz, H6′), 7.31 (1H, m, H-5′), 6.44 (1H, s, H-6), 5.75 (1H, d, J = 12.0 Hz, H-13a), 5.71 (2H, m, H-8, H-9), 5.64 (1H, d, J = 3.8 Hz, H-1), 5.20 (1H, t, J = 3.0 Hz, H-2), 4.94 (1H, d, J = 3.0 Hz, H-3), 4.92 (1H, brs, OH), 4.77 (1H, d, J = 13.1 Hz, H-15a), 4.67 (1H, d, J = 13.1 Hz, H15b), 3.81 (1H, d, J = 12.0 Hz, H-13b), 3.69 (1H, d, J = 12.0 Hz, H7′a), 3.09 (3H, s, H-2″), 2.99 (3H, s, H-3″), 2.75 (1H, m, H-7′b), 2.59 (1H, d, J = 2.9 Hz, H-7), 2.55 (1H, m, H-9′), 2.35 (3H, s, CH3COO), 2.19 (1H, m, H-8′a), 2.17 (3H, s, CH3COO), 2.04 (3H, s, CH3COO), 1.99 (3H, s, CH3COO), 1.89 (3H, s, CH3COO), 1.93 (1H, m, H-8′b), 1.73 (3H, s, Me-14), 1.62 (3H, s, Me-12), 1.25 (3H, d, J = 7.1 Hz, Me10′); 13C NMR (CDCl3, 100 MHz) δ 174.4 (C, C-11′), 170.2 (C, CH3COO), 169.8 (C, CH3COO), 169.7 (C, CH3COO), 169.4 (C, CH3COO), 168.7 (C, CH3COO), 167.0 (C, C-12′), 155.0 (C, C-1″), 154.3 (C, C-4′), 152.9 (CH, C-6′), 151.6 (CH, C-2′), 125.9 (CH, C5′), 124.7 (C, C-3′), 93.6 (C, C-5), 85.7 (C, C-11), 76.4 (CH, C-6), 75.6 (CH, C-3), 74.5 (CH, C-9), 73.1 (CH, C-8), 72.5 (CH, C-1), 70.5 (C, C-4), 69.8 (CH2, C-13), 68.9 (CH, C-2), 60.4 (CH2, C-15), 51.6 (C, C-10), 50.3 (CH, C-7), 38.3 (CH, C-9′), 36.8 (CH3, C-3″), 36.2 (CH3, C-4″), 34.3 (CH2, C-8′), 29.9 (CH2, C-7′), 23.3 (CH3, Me-12), 21.3 (CH3, CH3COO), 21.0 (CH3, CH3COO), 20.8 (CH3, CH3COO), 20.7 (CH3, CH3COO), 20.6 (CH3, CH3COO), 18.9 (CH3, Me-14), 17.9 (CH3, Me-10′); EIMS m/z 834 [M]+ (100), 775 (8), 746 (10), 703 (5), 684 (10), 671 (10), 644 (3), 614 (236), 572 (2), 509 (7); HREIMS m/z 834.3067 (calcd for C39H50N2O18, 834.3059). 6-Oxo-1α,2α,8β,9α,15-pentacetoxy-4β-hydroxy-3β,13-[4′-(3carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran (20). To a solution of 50 mg (0.066 mmol) of compound 13 in acetone (4 mL) in an ice bath was added the Jones reagent dropwise, until the solution turned orange. The reaction mixture was stirred for 1 h, and then 2-propanol was added. The solution was filtered through Florisil and washed with EtOAc, and the resulting residue was concentrated under reduced pressure and purified by preparative TLC using n-hexane−EtOAc (4:1) to give 31.1 mg (62%) of compound 20 as an amorphous, white solid: [α]20D −35.8 (c 0.5, CHCl3); UV (EtOH) λmax (log ε) 229 (1.58), 259 (3.43) nm; IR (neat) νmax 2929, 1753, 1593, 1371, 1226, 1161, 1118, 1049, 1003, 972, 934, 872, 843, 814, 758, 662, 631, 596, 494 cm−1; 1H NMR (CDCl3, 400 MHz) δ 9.15 (1H, s, H-2′), 8.69 (1H, d, J = 4.7 Hz, H-6′), 7.26 (1H, d, J = 5.1 Hz, H-5′), 5.58 (1H, d, J = 9.4 Hz, H-9), 5.74 (1H, d, J = 3.3 Hz, H-1), 5.70 (1H, dd, J = 3.8, 9.5 Hz, H-8), 5.28 (1H, t, J = 3.1 Hz, H-2), 5.03 (1H, d, J = 12.9 Hz, H-13a), 4.94 (1H, d, J = 3.0 Hz, H-3), 4.44 (1H, d, J = 12.9 Hz, H-13b), 4.33 (1H, d, J = 12.2 Hz, H-15a), 4.26 (1H, d, J = 12.2 Hz, H-15b), 3.98 (1H, brs, OH), 3.36 (1H, m, H-7′a), 2.88 (1H, m, H-7′b), 2.81 (1H, d, J = 3.7 Hz, H-7), 2.75 (1H, m, H-9′), 2.18 (3H, s, CH3COO), 2.15 (3H, s, CH3COO), 2.10 (1H, m, H-8′a), 2.08 (3H, s, CH3COO), 2.06 (3H, s, CH3COO), 1.96 (1H, m, H-8′b), 1.93 (3H, s, Me-12), 1.92 (3H, s, CH3COO), 1.75 (3H, s, Me-14), 1.32 (3H, d, J = 6.9 Hz, Me-10′); 13C NMR (CDCl3, 100 MHz) δ 200.6 (C, C-6), 174.1 (C, C-11′), 169.7 (C, CH3COO), 169.5 (C, CH3COO), 169.4 (C, CH3COO), 169.1 (C, CH3COO), 168.6 (C, C12′), 168.4 (C, CH3COO), 153.3 (CH, C-6′), 151.7 (CH, C-2′), 152.1 (C, C-4′), 124.3 (CH, C-5′), 123.1 (C, C-3′), 83.9 (C, C-5), 78.6 (C, C-11), 75.5 (CH, C-3), 74.3 (CH, C-9), 72.9 (CH, C-8), 70.1 (CH, C-1), 69.3 (C, C-4), 68.9 (CH2, C-13), 68.7 (CH, C-2), 59.8 (CH2, C-15), 54.3 (CH, C-7), 51.1 (C, C-10), 38.9 (CH, C-9′), 33.6 (CH2, C-8′), 30.8 (CH2, C-7′), 20.9 (CH3, Me-12), 20.8 (CH3, CH3COO), 20.7 (CH3, CH3COO), 20.6 (CH3, CH3COO), 20.5 (CH3, CH3COO), 20.4 (CH3, CH3COO), 19.0 (CH3, Me-14), 17.3 (CH3, Me-10′); EIMS m/z 761 [M]+ (6), 746 (48), 718 (28), 281 (18), 688 (54), 206 (15), 178 (21), 160 (15), 132 (13), 119 (12), 106 (11), 93 (100); HREIMS m/z 761.2545 (calcd for C36H43NO17, 761.2531). Anti-HIV Assays. Cellular viability was determined with the MTT method,34 and the viral infection was quantified by p24 antigen-based ELISA.35 Commercial AZT (Sigma; purity >98%) was used as a positive control, and the purity of the tested compounds was determined as >98%.

CH3COO), 1.86 (3H, s, CH3COO), 1.74 (3H, s, Me-14), 1.55 (3H, s, Me-12), 1.38 (1H, m, H-8′b), 1.09 (3H, d, J = 8.2 Hz, Me-10′); 13C NMR (CDCl3, 100 MHz) δ 174.2 (C, C-11′), 169.8 (C, CH3COO), 169.5 (C, CH3COO), 169.5 (C, CH3COO), 169.1 (C, CH3COO), 168.5 (C, C-12′), 166.7 (C, CH3COO), 156.9 (C, C-4′), 154.1 (C, C6″), 152.9 (CH, C-6′), 151.4 (CH, C-2′), 147.4 (CH, C-5″), 143.4 (CH, C-2″), 126.0 (CH, C-5′), 123.9 (C, C-3′), 120.2 (CH, C-3″), 112.2 (CH, C-4″), 93.5 (C, C-5), 85.8 (C, C-11), 75.3 (CH, C-3), 74.8 (CH, C-6), 74.1 (CH, C-9), 72.7 (CH, C-8), 72.2 (CH, C-1), 70.1 (CH2, C-13), 69.6 (C, C-4), 68.6 (CH, C-2), 60.2 (CH2, C-15), 51.3 (C, C-10), 49.9 (CH, C-7), 38.0 (CH, C-9′), 34.3 (CH2, C-8′), 29.4 (CH2, C-7′), 23.1 (CH3, Me-12), 21.0 (CH3, CH3COO), 20.8 (CH3, CH3COO), 20.6 (CH3, CH3COO), 20.5 (CH3, CH3COO), 20.4 (CH3, CH3COO), 18.7 (CH3, Me-14), 17.9 (CH3, Me-10′); EIMS m/z 857 [M]+ (78), 839 (36), 797 (23), 763 (74), 746 (100), 688 (16), 644 (12), 602 (5); HREIMS m/z 857.2708 (calcd for C41H47NO19, 857.2708). 1α,2α,8β,9α,15-Pentacetoxy-6β-lauroyloxy-4β-hydroxy-3β,13[4′-(3-carboxybutyl)]nicotinic acid-dicarbolactone-β-dihydroagarofuran (18). To 20.0 mg (0.026 mmol) of compound 13 dissolved in dry toluene (3.0 mL) were added 11.07 μL (3.0 equiv) of dry NEt3, 9.2 μL (1.5 equiv) of 2-lauroyl chloride, and a catalytic amount of 4(dimethylamino)pyridine. The reaction mixture was stirred for 48 h under reflux. Then, the solvent was eliminated under reduced pressure, and the resulting residue was purified by preparative TLC using nhexane−EtOAc (4:1) to give 14.9 mg (68%) of compound 18 as an amorphous, white solid: [α]20D −11.5 (c 0.3, CHCl3); UV (EtOH) λmax (log ε) 265 (3.36), 226 (3.92) nm; IR (neat) νmax 2926, 1751, 1591, 1461, 1370, 1227, 1159, 1094, 1047, 969, 907, 876, 762, 715, 593, 474 cm−1; 1H NMR (CDCl3, 400 MHz) δ 9.19 (1H, s, H-2′), 8.69 (1H, d, J = 5.0 Hz, H-6′), 7.28 (1H, s, H-5′), 6.64 (1H, s, H-6), 5.78 (1H, d, J = 12.0 Hz, H-13a), 5.69 (1H, d, J = 9.7 Hz, H-9), 5.63 (2H, m, H-1, H-8), 5.18 (1H, t, J = 3.0 Hz, H-2), 5.08 (1H, brs, OH), 4.93 (1H, d, J = 2.8 Hz, H-3), 4.71 (2H, s, H-15), 3.79 (2H, d, J = 12.0 Hz, H-13b, H-7′a), 2.71 (1H, m, H-7′b), 2.49 (1H, brs, H-7), 2.45 (3H, m, H-9′, H-2″), 2.33 (3H, s, CH3COO), 2.21 (1H, m, H-8′a), 2.16 (3H, s, CH3COO), 2.05 (3H, s, CH3COO), 2.00 (3H, s, CH3COO), 1.88 (3H, s, CH3COO), 1.77 (1H, m, H-8′b), 1.73 (3H, s, Me-14), 1.57 (3H, s, Me-12), 1.26 (18H, m, H-3″−H-11″), 1.23 (3H, d, J = 7.2 Hz, Me-10′), 0.88 (3H, t, J = 6.7 Hz, Me-12″); 13C NMR (CDCl3, 100 MHz) δ 174.4 (C, C-11′), 172.5 (C, C-1″), 170.1 (C, CH3COO), 169.8 (C, CH3COO), 169.7 (C, CH3COO), 169.3 (C, CH3COO), 168.7 (C, CH3COO), 166.6 (C, C-12′), 154.6 (C, C-4′), 153.3 (CH, C-6′), 151.8 (CH, C-2′), 126.1 (CH, C-5′), 124.7 (C, C3′), 93.9 (C, C-5), 85.9 (C, C-11), 75.5 (CH, C-3), 74.3 (CH, C-6), 74.3 (CH, C-9), 73.2 (CH, C-8), 72.5 (CH, C-1), 70.4 (CH2, C-13), 69.8 (C, C-4), 68.9 (CH, C-2), 60.4 (CH2, C-15), 51.5 (C, C-10), 50.1 (CH, C-7), 38.1 (CH, C-9′), 34.7 (CH2, C-2″), 34.5 (CH2, C-8′), 31.9 (CH2, C-3″), 31.8 (CH2, C-4″), 29.7 (CH2, C-7′), 29.7 (CH2, C-5″), 29.6 (CH2, C-6″), 29.3 (CH2, C-7″), 29.2 (CH2, C-8″), 29.1 (CH2, C9″), 24.5 (CH2, C-10″), 23.3 (CH3, Me-12), 22.7 (CH2, C-11′), 21.2 (CH3, CH3COO), 21.0 (CH3, CH3COO), 20.9 (CH3, CH3COO), 20.8 (CH3, CH3COO), 20.6 (CH3, CH3COO), 18.8 (CH3, Me-14), 18.2 (CH3, Me-10′), 14.1 (CH3, Me-12″); EIMS m/z 945 [M]+ (100), 930 (54), 887 (23), 873 (41), 872 (82), 842 (8), 765 (7), 749 (19), 748 (51), 746 (71), 704 (10), 688 (11), 644 (9), 602 (6); HREIMS m/z 945.4356 (calcd for C48H67NO18, 945.4358). 1α,2α,8β,9α,15-Pentacetoxy-6β-N,N-dimethylcarbamoyloxy-4βhydroxy-3β,13-[4′-(3-carboxybutyl)]nicotinic acid-dicarbolactoneβ-dihydroagarofuran (19). To 20.0 mg (0.026 mmol) of compound 13 dissolved in dry toluene (3.0 mL) were added 11.07 μL (3.0 equiv) of dry NEt3, 3.7 μL (3.0 equiv) of N,N-dimethylcarbamoyl chloride, and a catalytic amount of 4-(dimethylamino)pyridine. The reaction mixture was stirred for 24 h under reflux. Then, the solvent was eliminated under reduced pressure, and the resulting residue was purified by preparative TLC using a mixture of n-hexane−EtOAc (4:1) to give 19.3 mg (89%) of compound 19 as an amorphous, white solid: [α]20D −20.8 (c 0.2, CHCl3); UV (EtOH) λmax (log ε) 262 (2.69), 224 (3.23) nm; IR (neat) νmax 2928, 1751, 1591, 1553, 1458, 1371, 1227, 1180, 1099, 1044, 972, 911, 886, 758, 622, 597 cm−1; 1H NMR 1862



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ASSOCIATED CONTENT

S Supporting Information *

1 H and 13C NMR spectra of compounds 1−20. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 34 922318576. Fax: 34 922 318571. E-mail: aestebra@ull. es. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the Spanish MINECO (SAF 2012-37344-C03-C01) and the Spanish Ministerio de Educación (Programa Campus de Excelencia Internacional CEI10/00018). This project is also cofunded by the European Regional Development Fund (ERDF). We also thank EU Research Potential (FP7-REGPOT-2012-61367IMBRAIN) and Dr. M a A. Muñ o z (Laboratorio de Inmunobiologiá Molecular, Hospital General Universitario Gregorio Marañoń ) for carrying out the anti-HIV assays. F.G.N. thanks CajaCanarias-ULL for a predoctoral fellowship.

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REFERENCES

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