Tirucallane-Type Triterpenoids from the Fruit of Ficus carica and Their Cytotoxic Activity

Abstract

Nine new tirucallane-type triterpenoids, ficutirucins A–I (1–9), were isolated from the fruit of Ficus carica. Their structures were established on the basis of spectroscopic data and chemical methods. All isolates were evaluated for their cytotoxic activities against three human cancer cell lines, MCF-7, HepG-2, and U2OS. Compounds 1–3, 6, 7, and 9 exhibited moderate cytotoxic activities with IC₅₀ values of 11.67–45.61 µM against one or more of the three cancer cell lines.

Ficus (Moraceae) is a large genus which comprises about 800 species of trees, shrubs, hemiepiphytes, climbers, and creepers worldwide.¹⁾ Many species of this genus were used as folk medicines to treat cancer, pneumonia, diarrhea and cough in China.²⁾ Ficus carica LINN., an important member of this genus and commonly known as an edible fruit, was traditionally used to treat inflammation, diarrhea and indigestion.^3–5) Previous chemical studies of this plant had led to the isolation of phenolic acids, coumarins, and triterpenoids,^6–9) and the pharmacological investigations had demonstrated antioxidant, anticancer, and hepatoprotective activities.^6,10–12) In the search for bioactive metabolites from this medicinal plant, nine new tirucallane-type triterpenoids, ficutirucins A–I (1–9) (Fig. 1), were isolated from the fruit of Ficus carica. Herein, we reported the isolation and structure elucidation of these compounds, as well as their cytotoxic activities against MCF-7, HepG-2 and U2OS cell lines.

Fig. 1. Chemical Structures of Compounds 1–9

Results and Discussion

Ficutirucin A (1) was obtained as a white, amorphous powder. The molecular formula was determined to be C₃₂H₅₂O₅ by high resolution-electrospray ionization-mass spectra (HR-ESI-MS) (m/z 517.3884 [M+H]⁺, Calcd for C₃₂H₅₃O₅, 517.3888), indicating 7 degrees of unsaturation. The UV spectrum showed absorption maxima at 246 nm, typical for α,β-unsaturated carbonyl moiety.¹³⁾ The IR absorption bands at 3445 and 1636 cm⁻¹ implied the presence of hydroxyl and conjugated carbonyl groups, respectively. The ¹H-NMR spectrum of 1 (Table 1) showed signals due to one trisubstituted olefinic proton at δ_H 5.70 (1H, d, J=2.5 Hz), two oxygenated carbon protons at δ_H 4.47 (1H, dd, J=11.5, 4.0 Hz) and 3.34 (1H, dd, J=9.0, 4.0 Hz), two methines attaching to olefinic or carbonyl carbon at δ_H 2.73 (1H, m) and 2.20 (1H, s). In addition, it displayed an acetoxyl at δ_H 2.07 (3H, s), seven tertiary methyl singlets at δ_H 0.84, 0.88, 1.05, 1.17, 1.20, 1.20 and 1.22 (each 3H, s), and a secondary methyl at δ_H 0.90 (3H, d, J=6.0 Hz). In accordance with the molecular formula, 32 carbon signals were resolved in ¹³C-NMR spectra of 1 (Table 2), with aid of heteronuclear single quantum coherence (HSQC), assignable to nine methyls, eight methylenes, seven methines (two oxygenated and one olefinic), and eight quaternary carbons (two carbonyl, one olefinic and one oxygenated), these functionalities took 3 indices of hydrogen deficiency, and the remaining 4 indices suggested 1 to be tetracyclic. The aforementioned NMR data, with the rotating frame Overhauser enhancement spectroscopy (ROESY) cross-peak of H-20/H₃-18, indicated that compound 1 was a tirucallane-type triterpenoid,¹⁴⁾ and structurally similar to the brumollisol B,¹⁵⁾ except for the presence of two adjacent hydroxyl and an acetoxyl. The heteronuclear multiple bond connectivity (HMBC) correlations from the signals at H-24 (δ_H 3.34) to C-22 (δ_C 33.1), C-25 (δ_C 73.3), and C-26 (δ_C 23.5), from the methyl signals at H₃-26 (δ_H 1.17) and H₃-27 (δ_H 1.22) to C-25 indicated the two hydroxyl groups were linked to the C-24 and C-25, respectively. The location of the acetoxyl was deduced to be at C-3 by the HMBC cross-peak between H-3 (δ_H 4.47) and the acetyl carbonyl (δ_C 171.1) (Fig. 2). The relative configuration of 1 was determined on the basis of ROESY experiment. The correlations of H-3/H-5, H-5/H-9, H-9/H₃-18, and H₃-18/H-20 suggested the H-3, H-5, H-9, H₃-18, H-20 were cofacial and α-oriented, while the strong cross-peaks of H₃-19/H₃-30, H₃-30/H-17 revealed they were β-oriented (Fig. 2). The absolute configuration of C-24 was determined by the application of modified Mosher’s method.¹⁶⁾ The (R)-(+)-α-methoxy-trifluoromethylphenyl acetyl (MTPA) and (S)-(−)-MTPA esters of 1 were prepared. In the ¹H-NMR spectrum of (R)-(+)-MTPA ester of 1, signals assigned for H₃-18, H₃-21 were observed at a higher field than those in the (S)-(−)-MTPA ester, while signals due to H₃-26 and H₃-27 in the former ester were shifted to a lower field than those in the latter ester (Fig. 3). Therefore, the absolute configuration at C-24 of 1 was determined as 24R, and compound 1 was determined as (24R)-3β-acetoxytirucalla-7-ene-24,25-diol-6-one.

Table 1. ¹H-NMR Data (500 MHz) of Compounds 1–9 in CDCl₃ (δ in ppm, J in Hz)

Position	1	2	3	4	5	6	7	8	9
1a	1.70 m	1.69 m	1.82 m	1.81 m	1.95 m	1.96 m	1.68 m	1.85 m	1.86 m
1b	1.49 m	1.49 m	1.63 m	1.62 m	1.65 m	1.65 m	1.24 m	1.52 m	1.53 m
2a	1.68 m	1.68 m	1.75 m	1.76 m	1.85 m	1.86 m	1.67 m	1.79 m	1.80 m
2b	1.67 m	1.67 m	1.64 m	1.64 m	1.64 m	1.65 m	1.66 m	1.69 m	1.71 m
3	4.47 dd (11.5, 4.0)	4.47 dd (11.5, 4.5)	4.51 dd (11.0, 4.0)	4.51 dd (11.0, 4.0)	4.59 dd (10.5, 5.5)	4.59 dd (10.5, 5.5)	4.52 dd (12.0, 4.5)	4.53 dd (11.5, 4.5)	4.53 dd (11.5, 4.5)
5	2.20 s	2.20 s	1.37 dd (11.5, 4.5)	1.37 dd (11.5, 4.5)			1.42 m	1.76 dd (13.5, 5.5)	1.77 dd (13.5, 5.5)
6a			2.20 m	2.19 m	5.91 d (6.5)	5.91 d (6.0)	2.12 m	2.40 m	2.41 m
6b			2.07 m	2.07 m			1.97 m	2.32 m	2.34 m
7	5.70 d (2.5)	5.69 d (2.5)	5.34 br s	5.33 br s	5.50 d (6.0)	5.50 d (6.0)	5.25 d (3.5)
9	2.73 m	2.73 m					2.24 m
11a	1.73 m	1.74 m	5.20 br s	5.20 br s	5.37 m	5.37 m	1.53 m	2.38 m	2.38 m
11b	1.57 m	1.57 m					1.53 m	2.21 m	2.22 m
12a	1.88 m	1.87 m	2.15 m	2.14 m	2.25 m	2.25 m	1.81 m	1.76 m	1.77 m
12b	1.77 m	1.78 m	2.15 m	2.14 m	2.25 m	2.24 m	1.64 m	1.76 m	1.77 m
15a	1.77 m	1.77 m	1.64 m	1.64 m	1.69 m	1.70 m	1.51 m	2.12 m	2.11 m
15b	1.53 m	1.53 m	1.33 m	1.32 m	1.33 m	1.33 m	1.43 m	1.53 m	1.55 m
16a	2.03 m	2.02 m	1.97 m	1.96 m	2.02 m	2.01 m	1.79 m	1.98 m	2.01 m
16b	1.38 m	1.39 m	1.32 m	1.32 m	1.39 m	1.39 m	1.26 m	1.37 m	1.39 m
17	1.55 m	1.55 m	1.57 m	1.57 m	1.61 m	1.60 m	1.55 m	1.48 m	1.50 m
18	0.84 s	0.83 s	0.63 s	0.62 s	0.60 s	0.59 s	0.83 s	0.73 s	0.73 s
19	0.88 s	0.88 s	0.94 s	0.94 s	1.21 s	1.21 s	0.77 s	1.08 s	1.07 s
20	1.42 m	1.42 m	1.42 m	1.41 m	1.43 m	1.42 m	2.13 m	1.48 m	1.51 m
21	0.90 d (6.0)	0.91 d (6.5)	0.91 d (6.5)	0.92 d (6.5)	0.91 d (6.5)	0.92 d (6.5)	0.99 d (6.5)	0.91 d (6.0)	0.92 d (6.0)
22a	1.77 m	1.77 m	1.77 m	1.77 m	1.77 m	1.78 m	5.61 dd (15.5, 8.5)	2.20 m	2.28 m
22b	1.02 m	1.03 m	1.02 m	1.02 m	1.03 m	1.03 m		1.76 m	1.83 m
23a	1.59 m	1.58 m	1.59 m	1.59 m	1.60 m	1.60 m	5.44 dd (15.5, 7.0)	5.60 m	5.62 m
23b	1.15 m	1.15 m	1.16 m	1.15 m	1.16 m	1.16 m
24	3.34 dd (9.0, 4.0)	3.29 dd (10.0, 1.5)	3.35 t (6.5)	3.30 dd (10.0, 2.0)	3.35 t (6.5)	3.30 dd (10.0, 2.0)	3.85 d (7.0)	5.60 m	6.12 d (16.0)
26	1.17 s	1.16 s	1.17 s	1.17 s	1.17 s	1.17 s	1.15 s	1.31 s	1.84 s
27	1.22 s	1.22 s	1.22 s	1.22 s	1.22 s	1.22 s	1.20 s	1.31 s	4.86 s
28	1.20 s	1.20 s	0.87 s	0.87 s	1.09 s	1.08 s	0.85 s	0.88 s	0.88 s
29	1.20 s	1.20 s	0.96 s	0.96 s	1.19 s	1.19 s	0.93 s	0.96 s	0.96 s
30	1.05 s	1.05 s	0.84 s	0.84 s	0.96 s	0.96 s	0.97 s	0.96 s	0.97 s
3-OCOCH3	2.07 s	2.07 s	2.06 s	2.06 s	2.07 s	2.07 s	2.05 s	2.07 s	2.07 s

Table 2. ¹³C-NMR Data (125 MHz) of Compounds 1–9 in CDCl₃ (δ in ppm)

Position	1	2	3	4	5	6	7	8	9
1	36.8	36.8	35.5	35.5	35.2	35.2	37.0	34.5	34.5
2	23.5	23.4	24.4	24.4	24.3	24.3	24.4	24.0	24.1
3	80.8	80.8	81.1	81.1	78.9	78.9	81.3	79.9	79.9
4	37.1	37.1	38.1	38.1	40.4	40.4	38.0	37.9	37.9
5	65.4	65.4	48.6	48.6	150.4	150.4	51.0	48.6	48.6
6	199.5	199.5	23.6	23.6	118.5	118.5	24.0	35.7	35.7
7	125.1	125.1	118.4	118.4	114.6	114.6	118.0	197.9	197.9
8	170.7	170.7	141.6	141.6	140.7	140.7	145.8	139.2	139.2
9	50.5	50.5	144.9	145.0	145.4	145.4	49.0	165.2	165.2
10	43.7	43.7	36.2	36.2	39.5	39.5	35.0	39.3	39.3
11	17.8	17.8	115.7	115.7	120.0	120.0	18.2	23.8	23.8
12	32.9	32.9	38.5	38.5	39.4	39.4	33.7	29.9	30.0
13	43.2	43.2	44.3	44.3	45.0	45.0	43.8	44.8	44.9
14	52.4	52.4	49.7	49.7	49.5	49.5	51.4	47.8	47.8
15	33.0	33.1	31.4	31.4	30.9	30.9	34.2	31.6	31.7
16	27.9	27.8	28.1	28.1	28.1	28.0	28.7	28.7	28.8
17	52.9	52.7	51.3	51.2	51.2	51.1	52.7	48.7	48.9
18	22.0	22.0	16.3	16.3	16.4	16.4	22.2	15.7	15.7
19	14.5	14.5	20.9	20.9	30.7	30.7	13.3	18.9	18.9
20	35.9	36.4	36.2	36.7	36.2	36.7	40.5	36.8	37.2
21	18.4	18.7	18.5	18.8	18.5	18.8	20.2	18.9	19.1
22	33.1	33.4	33.3	33.7	33.3	33.7	141.1	39.2	39.9
23	28.6	28.9	28.6	29.0	28.6	29.0	126.2	125.5	129.5
24	78.8	79.7	78.9	79.8	78.9	79.8	79.8	139.8	134.4
25	73.3	73.4	73.3	73.4	73.3	73.4	73.0	70.9	142.4
26	23.5	23.5	23.5	23.5	23.5	23.5	23.9	30.2	18.9
27	26.8	26.7	26.8	26.7	26.9	26.9	26.5	30.1	114.2
28	28.3	28.3	27.8	27.8	26.7	26.7	27.7	27.4	27.4
29	16.1	16.1	16.6	16.6	26.3	26.3	16.0	16.3	16.3
30	25.1	25.1	23.3	23.3	22.6	22.6	27.4	24.4	24.5
3-OCOCH3	171.1	171.1	171.1	171.1	170.9	170.9	171.1	170.9	171.0
3-OCOCH3	21.4	21.4	21.4	21.4	21.5	21.5	21.4	21.3	21.3

Fig. 2. Key HMBC and ROESY of Compound 1

Fig. 3. Chemical Shift Differences (Δδ_H (S–R)) between (S)-MTPA and (R)-MTPA Esters of 1, 2 and 7

Ficutirucin B (2) gave the same molecular formula as compound 1 by HR-ESI-MS. The ¹H- and ¹³C-NMR spectra of 2 showed close resemblances to those of 1, except for slight differences of the ¹H- and ¹³C-NMR signals around C-24 (Tables 1, 2), suggesting 2 may be a C-24 epimer of 1,¹⁷⁾ which was further confirmed by the two dimensional (2D)-NMR (HSQC, HMBC, and ROESY) analysis. To confirm the spatial configuration of C-24 in 2, (S)- and (R)-MTPA esters of 2 also were synthesized. The Δδ (δ_S–δ_R) values for H₃-26 and H₃-27 (Δδ=0.01 and 0.04) were found to be positive, whereas those for the H₃-18 (Δδ=−0.02) and H₃-21 (Δδ=−0.04) were negative, which unequivocally demonstrated that 2 possesses 24S stereochemistry (Fig. 3). Thus the structure of 2 was deduced as (24S)-3β-acetoxytirucalla-7-ene-24,25-diol-6-one.

Ficutirucins C (3) and D (4) showed the same molecular formula, C₃₂H₅₂O₄, according to their HR-ESI-MS at m/z 523.3753 [M+Na]⁺ (Calcd for C₃₂H₅₂NaO₄, 523.3758) and 523.3757 [M+Na]⁺ (Calcd for C₃₂H₅₂NaO₄, 523.3758), respectively. The same to 1 and 2, both the 1D-NMR spectra of 3 and 4 also displayed the characteristic signals of an acetoxy, seven tertiary methyls, a secondary methyl, and two oxygenated carbons in their respective C-17 side chain. Analysis of the HSQC and HMBC spectra of these compounds was used to locate the acetoxy group at C-3, the two hydroxy groups at C-24 and C-25 as 1 and 2. In addition, compounds 3 and 4 have a Δ^7,9(11) conjugated diene [UV λ_max 232, 238 nm; IR 1640 cm⁻¹; δ_H 5.20 (1H, br s) and ca. 5.33 (1H, br s)],^18,19) which was further confirmed by the HMBC from H-7 (δ_H 5.33) to C-9 (δ_C 145.0), H-11 (δ_H 5.20) to C-8 (δ_C 141.6), H₃-19 (δ_H 0.94) to C-9, H₃-30 (δ_H 0.84) to C-8. The above evidence, together with the ROESY experiment, indicated that 3 and 4 possess a 24,25-dihydroxytirucall-Δ^7,9(11)-diene-3-ol acetate structure. The ¹³C-NMR chemical shift differences for the side chain made possible the stereochemical assignment at C-24 of 3 and 4.²⁰⁾ Thus, 24R and 24S epimers of 24,25-dihydroxy tirucallane exhibited ¹³C-NMR chemical shift differences (δ_S–δ_R) for the side chain as C-20 (δ_S–δ_R=0.5), C-21 (0.3), C-22 (0.4), C-23 (0.4), C-24 (0.9), and C-25 (0.1), C-26 (0), and C-27 (−0.1), which were almost consistent with those observed between 1 and 2: C-20 ((δ₂–δ₁=0.5), C-21 (0.3), C-22 (0.3), C-23 (0.3), C-24 (0.9), C-25 (0.1), C-26 (0), and C-27 (−0.1), as calculated from the ¹³C-NMR data in Table 2. Therefore, compound 3 was (24R)-3β-acetoxytirucalla-7,9(11)-diene-24,25-diol, while 4 was (24S)-3β-acetoxytirucalla-7,9(11)-diene-24,25-diol.

Ficutirucins E (5) and F (6) were found to share the same molecular formula, C₃₂H₅₀O₄, as deduced by HR-ESI-MS (m/z 499.3781 and 499.3784, [M+H]⁺ Calcd for C₃₂H₅₁O₄, 499.3782, respectively) and exhibited almost identical ¹³C-NMR spectra (Table 2), indicating these compounds to be another pair of epimers. The ¹H- and ¹³C-NMR data of compounds 5 and 6 (Tables 1, 2) were similar to those of 3 and 4, respectively, with the exception of an additional double bond at C-5 and C-6 in the ring B, which was proved by the HMBC correlations from the olefinic signal at H-6 (δ_H 5.91) to C-4 (δ_C 40.4), C-8 (δ_C 140.7) and C-10 (δ_C 39.5), and the characteristic UV spectra of conjugated triene (λ_max 306, 317 nm).²¹⁾ These indicated that compounds 5 and 6 were proposed to have the structure of 3β-acetoxytirucalla-5(6),7,9(11)-triene-24,25-diol, which was further supported from the analysis of their MS, HSQC, HMBC and ROESY spectra. Compounds 5 and 6 exhibited the signals of H-24 [5: δ_H 3.35 (t, J=6.5 Hz); 6: δ_H 3.30 (dd, J=10.0, 2.0 Hz)] and C-24 (5: δ_C 78.9; 6: δ_C 79.8), which were consistent with those of (24R) (1 and 3) and (24S) (2 and 4) epimers of 24,25-dihydroxytirucallanol (Tables 1, 2), respectively, indicating that 5 had a 24R configuration while 6 had a 24S configuration. In conclusion, compounds 5 and 6 were established to possess the structures of (24R)-3β-acetoxytirucalla-5,7,9(11)-triene-24,25-diol, and (24S)-3β-acetoxytirucalla-5,7,9(11)-triene-24,25-diol, respectively.

Ficutirucin G (7) exhibited an [M+Na]⁺ ion peak at m/z 523.3760 in the HR-ESI-MS, and its molecular formula was determined as C₃₂H₅₂O₄ (Calcd for C₃₂H₅₂NaO₄, 523.3758). Seven tertiary, one methyl doublet groups and an acetoxy group in the ¹H-NMR spectrum (Table 1), in addition to 32 carbons in ¹³C-NMR (Table 2) and the ROESY cross-peak of H-20/H₃-18, thus revealed the presence of a tirucallane acetate structure. Further analysis of 2D-NMR suggested 7 was an analogue of chisopanin M,²²⁾ except for replacement of the carbonyl by an O-acetyl group at C-3, which was further confirmed by the key HMBC correlations from H-3 (δ_H 4.52) to C-29 (δ_C 16.0), C-2 (δ_C 24.4) and carbonyl (δ_C 171.1). The coupling constant between H-22 and H-23 (J=15.5 Hz) defined the double bond to be in the E-configuration. The absolute configuration at C-24 was decided to be 24S by the modified Mosher’s method (Fig. 3), hence, 7 was (24S)-3β-acetoxytirucalla-7,22-diene-24,25-diol.

Ficutirucin H (8) was obtained as a white, amorphous powder. The molecular formula was determined to be C₃₂H₅₀O₄ by HR-ESI-MS (m/z 499.3780 [M+H]⁺ Calcd for C₃₂H₅₁O₄, 499.3782). The UV spectrum displayed an absorption maximum at 253 nm. The ¹H- and ¹³C-NMR spectra (Tables 1, 2), with the aid of HSQC, exhibited resonances for a bisubstituted double bond (δ_H 5.60, 2H, m; δ_C 125.5 and 139.8), an α,β-unsaturated ketone (δ_C 197.9, 139.2, and 165.2), in addition to an acetoxy group, seven tertiary and one secondary methyl groups. The ring carbon signals in the ¹³C-NMR spectra were almost identical with those of dihydrolanosterol acetate,²³⁾ suggesting that they had the same tetracarbocyclic skeleton. Furthermore, ¹H-NMR chemical shift (δ_H 0.91, d, J=6.0 Hz) of Me-21 along with the ROESY correlation of H-20 and H₃-18 were consistent with those of a tirucallane triterpenes. On the other hand, the deshielded nature of the methyls (δ_H 1.31, s, 6H) indicated they must be attached to the fully substituted oxygenated carbon (δ_C 70.9), which must carry the tertiary hydroxyl group.¹⁹⁾ The HMBC correlations from H₃-26 (δ_H 1.31) and H₃-27 (δ_H 1.31) to C-25 (δ_C 70.9), and from H-23 (δ_H 5.60) and H-24 (δ_H 5.60) to C-25 and C-22 (δ_C 39.2) confirmed the side chain to be [CH₂–CH=CH–C(CH₃)₂OH], which was same to terminal side chain of kansenonol.¹⁹⁾ Moreover, the superimposable ¹H- and ¹³C-NMR signals of compound 8 due to the terminal side chain (C22–C27) to those of kansenonol supported the E-configuration of the olefinic bond. Therefore, the structure of 8 was established as (23E)-3β-acetoxytirucalla-8,23-diene-25-ol-7-one.

Ficutirucin I (9) was obtained as a white, amorphous powder. The molecular formula was determined to be C₃₂H₄₈O₃ by HR-ESI-MS (m/z 481.3674 [M+H]⁺ (Calcd for C₃₂H₄₉O₃, 481.3676), 18 mass units less than 8. The ¹H and ¹³C signals (Tables 1, 2) arising from triterpene ring systems of 9 were almost indistinguishable from those of the corresponding signals of 8, suggesting that they possess the same tetracyclic moiety. The ¹H-NMR signals of olefinic protons at δ_H 4.86 (2H, s, H₂-27), 5.62 (1H, m, H-23) and 6.12 (1H, d, H-24), suggested the presence of the 23,25-diene in the side chain,²⁴⁾ which was supported by HMBC correlations for H₂-27 (with C-24 and C-26), H₃-26 (with C-24, C-25, and C-27), H-23 (with C-22 and C-25) and H-24 (with C-22, C-25, C-26 and C-27). The coupling constant between H-23 and H-24 (J=16.0 Hz) supported the E-configuration of the olefinic bond, which was confirmed by the ROESY cross-peak of H-23/H₃-26 and H-24/H₂-27. Therefore, the structure of 9 was established as (23E)-3β-acetoxytirucalla-8,23,25-triene-7-one.

The fruit of Ficus carica has been reported to have the anticancer effect.^25–29) However, the bioactive compounds responsible for antitumor remained elusive. Since cytotoxic activities of tirucallane-type triterpenoids had been reported,^15,30) we further examined the cytotoxic activities of compounds 1–9 using three human cancer cell lines by MTT assay. As shown in Table 3, six compounds 1, 2, 3, 6, 7 and 9 displayed moderate cytotoxic activities with IC₅₀ values of 11.67–45.61 µM against more than one cancer cell lines. Among them, 1 exhibited the highest activity with an IC₅₀ value of 11.67 µM against HepG-2, 2 revealed cytotoxic activities against MCF-7 and HepG-2 cell lines with the IC₅₀ values of 17.94 µM and 17.76 µM, and 6 showed activity against U2OS of 20.70 µM. Additionally, it was found that, compounds 1 and 2 showed the higher activities against MCF-7 and HepG-2 cell lines than compounds 3–7, indicating that the presence of the α,β-unsaturated ketone moiety at C-6, C-7 and C-8 might play an important role in the cytotoxicities. Compounds 2 and 6 with the 24S configurations exhibited the stronger activities on U2OS cell lines than their 24R-epimers, 1 and 5. For compounds 8 and 9, it seemed that the 23,25-diene in the side chain might increase the inhibitory activities.

Table 3. The Cytotoxic Activities of the Isolates from F. carica^a)

Compounds	MCF-7	HepG-2	U20S
1	27.78±2.21	11.67±0.09	34.93±3.28
2	17.94±0.33	17.76±0.06	27.66±0.17
3	>50c)	42.02±1.73	>50
4	>50	>50	>50
5	>50	>50	>50
6	45.61±1.34	24.68±5.11	20.70±1.99
7	27.48±0.57	>50	28.08±0.26
8	>50	>50	>50
9	31.28±1.14	23.19±1.13	32.76±1.00
cis-Platinumb)	9.69±0.26	8.30±0.58	6.19±0.13

a) Results were expressed as IC₅₀ values in µM (n=3). b) Positive controls. c) >50 µM was inactive.

In this paper, nine new tirucallane-type triterpenoids were isolated from the fruit of Ficus carica. Among them, seven compounds had the 24,25-diol moiety and three pairs of 24-epimers were found. The absolute configurations of the epimers were determined by the modified Mosher’s method and comparison of spectra data. Six compounds showed cytotoxicities on one or more cell lines, which indicated the tirucallane-type triterpenoids may contribute to the anticancer effect of this medicinal plant.

Experimental

General Experimental Procedures

Optical rotations were measured with a JASCO P-1020 polarimeter. UV spectra were recorded on a Shimadzu UV-2450 spectropolarimeter. IR spectra were measured in KBr-disc on a Bruker Tensor 27 spectrometer. NMR spectra were obtained on a Bruker AV-500 (or AV-300) NMR instrument at 500 (or 300) MHz (¹H) and 125 MHz (¹³C) with tetramethylsilane (TMS) as the internal standard. HR-ESI-MS was carried out on an Agilent UPLC-Q-TOF (6520B). Column chromatography (CC) was performed on D101 macroporous resion (The Chemical Plant of Nankai University), silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Co., Ltd.), ODS (40–63 µm, FuJi), and Sephadex LH-20 (Pharmacia). Preparative HPLC was carried out using a Shimadzu LC-6A instrument with a SPD-20 A detector using a Shim-pack PRC-ODS column (20×250 mm, i.d.) and a Shimadzu CBM-20 A recycling-preparative HPLC system with a SPD-20 A detector using a Shim-pack PRC-ODS (H) column (20×250 mm, i.d.). Analytical HPLC was measured on an Agilent 1260 Series instrument with a DAD detector using a Shim-pack VP–ODS column (4.6×250 mm, i.d.).

Plant Material

The unriped fruit of Ficus carica was collected from Shandong Province, China, in November 2012, and authenticated by Professor Mian Zhang, Department of Pharmacognosy, China Pharmaceutical University. A voucher specimen (No. FC-201211) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.

Extraction and Isolation

The air-dried fruit of F. carica (15 kg) was extracted with 95% EtOH (3×30 L) under reflux, and then concentrated under vacuum to yield a crude extract (2.5 kg). The extract was subjected to a D101 macroporous resion column, eluting with a gradient of EtOH–H₂O (40 : 60, 70 : 30, 90 : 10, v/v), to afford three fractions (Fr. A–C). Fraction C (EtOH–H₂O, 90 : 10, 438 g) was suspended in MeOH–H₂O (50 : 50) and partitioned successively with petroleum ether and methylene dichloride. The petroleum ether portion (Fr. CP, 235 g) was subjected to silica gel CC using petroleum ether–acetone eluent in gradient (1 : 0, 200 : 1, 100 : 1, 50 : 1, 25 : 1, 10 : 1 and 5 : 1) to yield nine subfractions (Fr. CPA–CPI). Fraction CPI was further purified by silica gel using petroleum ether–acetone (15 : 1, 7 : 1 and 3 : 1) to get five subfractions (Fr. CPI1–CPI5). Fraction CPI2 was then separated by silica gel CC, Sephadex LH-20 and ODS column, then finally purified by preparative HPLC (CH₃CN–H₂O, 65 : 35) to produce 8 (4.1 mg) and 9 (5.3 mg). Fraction CPI3 was separated over silica gel CC (petroleum ether–acetone, 8 : 1, 4 : 1) to give four subfractions (Fr. CPI3a–CPI3d). Fraction CPI3c was subjected to octadecyl silica (ODS) column chromatography eluted with MeOH–H₂O (70 : 30, 80 : 20 and 90 : 10) to yield seven fractions (Fr. CPI3c1–CPI3c7). Fraction CPI3c5 was chromatographed on silica gel with a mixture of petroleum ether–acetone (10 : 1, 5 : 1) to give ten subfractions (Fr. CPI3c5a–CPI3c5j). Fraction CPI3c5f was subjected to ODS column chromatography (MeOH–H₂O, 70 : 1, 80 : 1, 90 : 1) and preparative HPLC (MeOH–H₂O, 90 : 10) to yield Fractions CPI3c5f4c1–CPI3c5f4c2. Fraction CPI3c5f4c1 subsequently separated by recycling-preparative HPLC using MeOH–H₂O (85 : 15) to afford 6 (3.4 mg) and 5 (3.9 mg). Fraction CPI3c5f4c2 then separated by recycling-preparative HPLC using MeOH–H₂O (90 : 10) to afford 4 (3.3 mg), 3 (3.5 mg) and 7 (5.0 mg). Fraction CPI4 was subjected to ODS column chromatography eluted with MeOH–H₂O (70 : 30, 80 : 20 and 90 : 10) to yield eight fractions (Fr. CPI4a–CPI4h). Fraction CPI4b was subjected to ODS column and Sephadex LH-20 column, then purified via preparative HPLC using MeOH–H₂O (80 : 20) to yield Fraction CPI3c5f4c2b (17 mg), finally separated by recycling-preparative HPLC using CH₃CN–H₂O (65 : 35) to afford 1 (3.1 mg) and 2 (4.2 mg).

Ficutirucin A (1): White, amorphous powder; [α]_D²⁵ −12.8° (c=0.11, MeOH); UV (MeOH) λ_max (log ε) 246 (3.73); IR (KBr) ν_max 3445, 2958, 2924, 1636, 1385 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z 517.3884 [M+H]⁺ (Calcd for C₃₂H₅₃O₅, 517.3888).

Ficutirucin B (2): White, amorphous powder; [α]_D²⁵ −3.4° (c=0.10, MeOH); UV (MeOH) λ_max (log ε) 246 (3.76) nm; IR (KBr) ν_max 3450, 2960, 2925, 1640, 1400 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z 517.3889 [M+H]⁺ (Calcd for C₃₂H₅₃O₅, 517.3888).

Ficutirucin C (3): White, amorphous powder; [α]_D²⁵ −59.6° (c=0.12, MeOH); UV (MeOH) λ_max (log ε) 232 (3.57), 238 (3.62) nm; IR (KBr) ν_max 3474, 2968, 1640, 1401 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z 523.3753 [M+Na]⁺ (Calcd for C₃₂H₅₂NaO₄, 523.3758).

Ficutirucin D (4): White, amorphous powder; [α]_D²⁵ −58.6° (c=0.09, MeOH); UV (MeOH) λ_max (log ε) 232 (3.68), 238 (3.72) nm; IR (KBr) ν_max 3452, 2970, 2926, 1639, 1386 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z 523.3757 [M+Na]⁺ (Calcd for C₃₂H₅₂NaO₄, 523.3758).

Ficutirucin E (5): White, amorphous powder; [α]_D²⁵ −27.5° (c=0.11, MeOH); UV (MeOH) λ_max (log ε) 204 (3.34), 306 (2.87), 317 (2.87) nm; IR (KBr) ν_max 3474, 2961, 1715, 1638, 1385, 1066 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z 499.3781[M+H]⁺ (Calcd for C₃₂H₅₁O₄, 499.3782).

Ficutirucin F (6): White, amorphous powder; [α]_D²⁵ −51.1° (c=0.10, MeOH); UV (MeOH) λ_max (log ε) 205 (3.32), 306 (2.77), 317 (2.77) nm; IR (KBr) ν_max 3450, 2970, 2926, 1715, 1639, 1384, 1246, 1034 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z 499.3784 [M+H]⁺ (Calcd for C₃₂H₅₁O₄, 499.3782).

Ficutirucin G (7): White, amorphous powder; [α]_D²⁵ −24.0° (c=0.09, MeOH); UV (MeOH) λ_max (log ε) 204 (3.35), 239 (2.61) nm; IR (KBr) ν_max 3455, 2962, 2924, 1639, 1399 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z 523.3760 [M+Na]⁺ (Calcd for C₃₂H₅₂NaO₄, 523.3758).

Ficutirucin H (8): White, amorphous powder; [α]_D²⁵ −3.23° (c=0.10, MeOH); UV (MeOH) λ_max (log ε) 202 (3.04), 253 (3.28) nm; IR (KBr) ν_max 3450, 2959, 2923, 1713, 1639, 1475, 1384, 1251 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z 499.3780 [M+H]⁺ (Calcd for C₃₂H₅₁O₄, 499.3782).

Ficutirucin I (9): White, amorphous powder; [α]_D²⁵ −4.8° (c=0.09, MeOH); UV (MeOH) λ_max (log ε) 201 (3.46), 254 (3.51) nm; IR (KBr) ν_max 3450, 2970, 2926, 1750, 1644, 1463, 1384, 1246, 1029 cm⁻¹; ¹H- and ¹³C-NMR data, see Tables 1 and 2; HR-ESI-MS m/z 481.3674 [M+H]⁺ (Calcd for C₃₂H₄₉O₃, 481.3676).

Preparation of the (S)- and (R)-MTPA Ester Derivatives of 1, 2 and 7

Dimethylaminopyridine (DMAP, 2 mg) and (S)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) 4 µL were added to a solution of 1 (2 or 7) (1 mg) in dried pyridine (50 µL), and then the mixture was allowed to stand overnight at 30°C. MeOH (2 mL) was added to quench the reaction and finally purified by preparative HPLC using MeOH–H₂O (88 : 12 or 95 : 5) to give pure (S)-MTPA ester of 1 (2 or 7) (0.9 mg). The (R)-MTPA ester of 1 (2 or 7) was prepared using the method described above.

(R)-MTPA (1R) and (S)-MTPA (1S) Esters of 1. 1R: ¹H-NMR (CDCl₃, 500 MHz) δ: 0.79 (3H, d, J=6.0 Hz, H₃-21), 0.80 (3H, s, H₃-18), 0.88 (3H, s, H₃-19), 1.04 (3H, s, H₃-30), 1.16 (3H, s, H₃-26), 1.20 (3H, s, H₃-28), 1.20 (3H, s, H₃-29), 1.22 (3H, s, H₃-27), 2.07 (3H, s, –OCOCH₃), 2.20 (1H, s, H-5), 2.72 (1H, m, H-9), 4.47 (1H, dd, J=11.5, 4.0 Hz, H-3), 4.96 (1H, dd, J=10.0, 2.0 Hz, H-24), 5.70 (1H, d, J=2.5 Hz, H-7). 1S: ¹H-NMR (CDCl₃, 500 MHz) δ: 0.82 (3H, s, H₃-18), 0.85 (3H, d, J=6.0 Hz, H₃-21), 0.88 (3H, s, H₃-19), 1.04 (3H, s, H₃-30), 1.15 (3H, s, H₃-26), 1.18 (3H, s, H₃-27), 1.20 (3H, s, H₃-28), 1.20 (3H, s, H₃-29), 2.07 (3H, s, –OCOCH₃), 2.20 (1H, s, H-5), 2.72 (1H, m, H-9), 4.47 (1H, dd, J=11.5, 4.0 Hz, H-3), 4.98 (1H, dd, J=9.5, 2.5 Hz, H-24), 5.69 (1H, d, J=3.0 Hz, H-7).

(R)-MTPA (2R) and (S)-MTPA (2S) Esters of 2. 2R: ¹H-NMR (CDCl₃, 500 MHz) δ: 0.81 (3H, s, H₃-18), 0.89 (3H, d, J=7.0 Hz, H₃-21), 0.88 (3H, s, H₃-19), 1.04 (3H, s, H₃-30), 1.15 (3H, s, H₃-26), 1.18 (3H, s, H₃-27), 1.20 (3H, s, H₃-28), 1.20 (3H, s, H₃-29), 2.07 (3H, s, –OCOCH₃), 2.20 (1H, s, H-5), 2.72 (1H, m, H-9), 4.47 (1H, dd, J=11.0, 3.0 Hz, H-3), 4.95 (1H, dd, J=9.5, 1.5 Hz, H-24), 5.69 (1H, d, J=2.5 Hz, H-7). 2S: ¹H-NMR (CDCl₃, 500 MHz) δ: 0.79 (3H, s, H₃-18), 0.85 (3H, d, J=6.5 Hz, H₃-21), 0.88 (3H, s, H₃-19), 1.03 (3H, s, H₃-30), 1.16 (3H, s, H₃-26), 1.20 (3H, s, H₃-28), 1.20 (3H, s, H₃-29), 1.22 (3H, s, H₃-27), 2.07 (3H, s, –OCOCH₃), 2.20 (1H, s, H-5), 2.71 (1H, m, H-9), 4.47 (1H, dd, J=11.5, 4.0 Hz, H-3), 4.95 (1H, dd, J=9.5, 2.0 Hz, H-24), 5.69 (1H, d, J=2.5 Hz, H-7).

(R)-MTPA (7R) and (S)-MTPA (7S) Esters of 7. 7R: ¹H-NMR (CDCl₃, 300 MHz) δ: 0.76 (3H, s, H₃-19), 0.82 (3H, s, H₃-18), 0.85 (3H, s, H₃-28), 0.93 (3H, s, H₃-29), 0.95 (3H, s, H₃-30), 0.99 (3H, d, J=6.0 Hz, H₃-21), 1.15 (3H, s, H₃-26), 1.15 (3H, s, H₃-27), 2.06 (3H, s, –OCOCH₃), 5.45 (1H, dd, J=15.0, 9.0 Hz, H-23), 5.78 (1H, dd, J=15.0, 9.0 Hz, H-22). 7S: ¹H-NMR (CDCl₃, 300 MHz) δ: 0.76 (3H, s, H₃-19), 0.80 (3H, s, H₃-18), 0.85 (3H, s, H₃-28), 0.93 (3H, s, H₃-29), 0.96 (3H, s, H₃-30), 0.96 (3H, d, J=4.5 Hz, H₃-21), 1.18 (3H, s, H₃-26), 1.21 (3H, s, H₃-27), 2.06 (3H, s, –OCOCH₃), 5.32 (1H, dd, J=15.0, 9.0 Hz, H-23), 5.69 (1H, dd, J=15.0, 9.0 Hz, H-22).

Cytotoxic Assay

The cytotoxic activity was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method using cis-platinum as positive control according to the published paper.³¹⁾ MCF-7 (Human breast adenocarinoma cell line), HepG-2 (human hepatoma cell line), and U2OS (human osteosarcoma cell line) were cultured on RPMI-1640 medium supplemented with 10% foetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C with 5% CO₂, then dispensed into 96-well plates at 5×10³ cells per well in 200 µL medium at 37°C for 24 h. Compounds 1–9 were added at different concentrations (0–50 µM). After treatment for 48 h, 20 µL of MTT solution (5 mg/mL) was added and cultured for 4 h. Then the supernatant was discarded and dimethyl sulfoxide (DMSO) was added (150 µL/well). Absorbance was measured at 570 nm by a Universal Microplate Reader.

Acknowledgments

This research work was financially supported by the National Key Scientific and Technological Special Projects (2012ZX09103-101-007), New Century Excellent Talents in University (NCET-12-09-77), Program for Changjiang Scholars and Innovative Research Team in University (IRT1193) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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