Isolation and Biological Evaluation of Jatrophane Diterpenoids from Euphorbia dendroides
Ivana S. Aljani,*,?,# Milica Pei,?,# Slobodan M. Milosavljevi, Nina M. Todorovi,? Milka Jadranin,? cc sc c c Goran Milosavljevi,^ Dragan Povrenovi,|| Jasna Bankovi,? Nikola Tani,? Ivanka D. Markovi,3 c c c c c Sabera Rudiji,? Vlatka E. Vajs,? and Vele V. Teevi*,O z c s c
Institute of Chemistry, Technology and Metallurgy, Center for Chemistry, University of Belgrade, Njegoeva 12, 11001 Belgrade, Serbia s Institute for Biological Research, Department of Neurobiology, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia Serbian Academy of Sciences and Arts, Knez Mihailova 35, 11001 Belgrade, Serbia ^ Konekta Konsalting, Bulevar Cara Lazara 29/119, 21000 Novi Sad, Serbia Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia 3 Faculty of Medicine, University of Belgrade, Doktora Subotia 12, 11000 Belgrade, Serbia c O Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11158 Belgrade, Serbia
S b Supporting Information
ABSTRACT: From the Montenegrin spurge Euphorbia dendroides, seven new diterpenoids [jatrophanes (1?6) and a tigliane (7)] were isolated and their structures elucidated by spectroscopic techniques. The biological activity of the new compounds was studied against four human cancer cell lines. The most e?ective jatrophane-type compound (2) and its structurally closely related derivative (1) were evaluated for their interactions with paclitaxel and doxorubicin using a multidrug-resistant cancer cell line. Both compounds exerted a strong reversal potential resulting from inhibition of P-glycoprotein transport.
uphorbia dendroides L. (Euphorbiaceae) is a small tree distributed in the Mediterranean region. Recent studies have shown macrocyclic jatrophane diterpenoids from Euphorbia species as a new structural class of inhibitors of P-gp.1?3 As part of an investigation of spurges from the southeastern Balkan region, a jatrophane diterpenoid fraction was puri?ed from E. dendroides. Isolation and structure determination work yielded six new jatrophanes (1?6) and a new tigliane (7). Two of the jatrophanes (1 and 2) were shown to behave as potent multi-drug-resistant (MDR) modulators, each reversing resistance to paclitaxel in NCI-H460/R cells (a MDR cancer cell line).4 The sensitivity of NCI-H460/R cells to another anticancer chemotherapeutic agent, doxorubicin, was also investigated in the presence of these jatrophanes. Described herein are the isolation, structure elucidation, and biological evaluation of compounds 1?7.
RESULTS AND DISCUSION The exhaustive extraction of a lyophilized aqueous ethanol extract of E. dendroides with hexane, followed by application of several preparative chromatographic techniques, a?orded six new jatrophanes, euphodendrophanes A?F (1?6), and the tigliane euphodendriane A (7).
Copyright r XXXX American Chemical Society and American Society of Pharmacognosy
Euphodendrophane A (1) was obtained as a colorless, amorphous solid. The molecular formula, C37H49NO12, was determined on the basis of an ion at m/z 700.3335 (calcd 700.3327) in the HRESIMS. The structural elucidation of 1 was achieved with the use of 1D and 2D NMR spectroscopy, as well as comparison of the NMR spectra obtained for jatrophane polyesters isolated previously from E. dendroides5 and other Euphorbia species, possessing similar acylation patterns, namely, E. turczaninowii,6 E. obtusifolia,7 E. terracina,8 E. amygdaloides,9 and E. altotibetic.10 The overall NMR characteristics were suggestive of 1 being a diterpenoid bearing ?ve ester functionalities. The NMR data (Tables 1 and 2) revealed the nature of the ester moieties as two acetates, a propionate, an isobutyrate, and a nicotinate. According to its 13C and 1H NMR spectra, compound 1 was found to contain six oxygenated sp3 carbons (?ve secondary and one tertiary), with ?ve of these bearing ester groups and the remaining one a hydroxy group (H 2.81 s, exchangeable with D2O). The NMR spectra also showed a signal for a keto carbonyl (C 214.7), two double bonds (one exocyclic and one trans-disubstituted), an aliphatic methylene, four methyls (two 1H NMR
Received: March 18, 2011
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Journal of Natural Products doublets and two singlets), and a sp3 quaternary carbon (C 40.6). This, in combination with the molecular formula, corresponding to 14 double-bond equivalents, indicated a bicyclic diterpene skeleton. Chart 1
The molecular framework of 1 was established by the application of 2D NMR techniques (COSY, HMBC, and NOESY). Thus, the observed COSY correlations (denoted in parentheses) enabled identi?cation of three independent proton coupling networks: A (H2-1/H-2, H-2/H3-16, H-2/H-3, H-3/H-4, and H-4/H-5), B (H-7/H-8 and H-8/H-9), and C (H-11/H-12, H-12/H-13, and H-13/H3-20) denoted with bold lines in Figure 1. The 2,3JC?H correlations inferred from the HMBC spectrum allowed the connections of A, B, and C to be established (Figure 1). Thus, the long-range correlations of H-5 and H-7 with C-6 and C-17 (exocyclic double bond) indicated the connection of A and B via the ole?nic C-6. HMBC cross-peaks of protons attributed to the gem-dimethyl groups H319 and H3-18 (positioned at C-10) with C-9, C-10, and C-11 de?ned the connection between B and C, through C-10. Finally, a HMBC correlation of the ketone carbonyl (C-14) with H-13, H3-20, and the H2-1 methylene revealed the linkage of C and A. This evidence led to the diterpenoid carbon framework of 1 being proposed as a bicyclo[10.3.0]pentadecane with 2,10,10,13tetramethyl-6-exo-methylene, commonly known as a jatrophane skeleton, with oxygenated carbons at positions 3, 5, 7, 8, 9, 14
Table 1. 1H NMR Data for Compounds 1?5 [500 MHz, CDCl3, TMS, (ppm) (J = Hz)]
position 1R 1 2 3 4 5 7 8 9 11 12 13 16 17a 17b 18 19 20 OR1-3 1 2.51 dd (14; 11) 1.59 dd (14; 11) 2.26 m 5.55 t (4.5) 3.13 brs 5.43 brs 5.08 brs 5.63 brs 5.22 brs 5.92 d (15.5) 5.71 m 4.25 0.93 d (6.5) 5.06 brs 5.14 brs 1.08 brs 1.33 brs 1.22 d (7.02) 2.41 dq (7.5; 4.5) 1.19 t (7.5) OR2-5 OR3-7 2.07 s 1.95 bm 0.86 brs 0.65 brs OR4-8 1.98 s 2 2.53 dd (14; 11) 1.60 dd (14; 11) 2.26 m 5.55 t (4.5) 3.13 brs 5.41 brs 5.07 brs 5.68 brs 5.22 brs 5.92 d (15.5) 5.71 m 4.25 0.93 d (6.5) 5.05 brs 5.14 brs 1.07 brs 1.34 brs 1.23 d (7.02) 2.64 h (7.0) 1.27 (7.0) 1.22 (7.0) 2.08 s 1.98 bm 0.86 d (7.0) 0.63 d (7.0) 1.98 2.03 s 2.49 h (7.5) 0.93d (7.0) 0.73 brs 2.10 s 8.01 d (8.5) 7.54 t (7.5) 7.42 (8.0) OR5-9 9.25 brs 8.80 brs 8.26 brd 7.45 brdd OR6-15 2.81 s 9.26 brs 8.82 brs 8.26 brd 7.47 brdd 2.81 s
3 3.06 dd (14; 6.5) 1.64 bt (14) 2.29 m 5.41 bs 2.79 d (3.0) 5.83 brs 5.08 brs 5.53 brs 5.21 d (2) 5.94 d (16) 5.76 m 3.61 m 0.92 d (6.5) 5.06 brs 5.09 brs 1.06 brs 1.25 brs 1.20 d (6.0) 2.40 dq (7.5; 4.5) 1.19 d (7.5)
4 2.51 brs 1.58 dd 2.25 brs 5.57 brs 3.12 brs 5.47 brs 4.88 brs 5.65 brs 5.06 brs 5.90 d (16.0) 5.83 m 4.20 m 0.93 d (6.5) 5.01 brs 5.08 brs 0.98 brs 1.39 brs 1.22 d (7.0) 2.60 h (7.0) 1.26 d (7.0) 1.19 d (7.0) 2.09 s 2.07 s
5 2.40 m 1.60 t (12) 2.29 bm 5.60 brs 3.07 brs 5.42 brs 4.82brs 5.70 brs 5.06 brs 5.86 d (16.0) 5.92 m 4.21 m 0.93 d (6.5) 4.96 brs 5.03 brs 0.96 brs 1.42 brs 1.16 d (7.0) 2.40 q (7.5) 1.17 t (7.5) 2.09 s 2.06 h (7.0) 1.20 d (7.0) 1.16 d (7.0) 8.02 d (8.5) 7.56 t (7.5) 7.43 (8.0) 1.97 s
9.21 brs 8.81 brs 8.20 brd 7.45 brdd 2.12 s
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Journal of Natural Products Table 2. 13C NMR Data for Compounds 1?5 [(125 MHz, CDCl3, TMS, (ppm)]
position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OR1-3 10 20 30 40 5-R2 100 200 OR3-7 1000 2000 3000 4000 OR4-8 10000 20000 30000 40000 50000 OR5-9 100000 200000 300000 400000 500000 600000 OR6-15 1000000 2000000
1 49.9 379 77.7 53.8 68.0 144.9 69.0 70.8 81.4 40.6 137.6 127.7 40.8 214.7 87.7 13.7 113.6 25.2 24.9 18.6 173.1 27.7 9.1
2 50.0 38.0 77.6 53.7 68.0 144.8 69.1 70.8 81.4 40.9 137.6 127.8 40.6 214.6 87.9 13.7 113.8 25.3 24.8 18.6 175.6 34.3 19.8 18.5
3 45.8 38.3 76.2 52.8 67.8 144.7 68.4 71.1 80.0 40.2 137.0 131.5 42.8 212.3 92.9 13.4 111.1 26.9 24.0 19.9 173.0 28.0 9.2
4 50.0 38.2 77.5 53.6 68.8 145.5 69.7 71.2 80.7 40.5 137.8 127.1 39.9 214.7 87.5 13.6 111.8 26.3 23.8 18.2 175.5 34.4 18.6 19.7
5a 49.7 38.3 77.3 53.9 68.7 145.9 69.0 71.3 80.6 40.6 137.7 127.0 39.8 215.1 87.1 13.5 110.9 26.6 23.4 18.3 173.2 27.8 9.3
Figure 1. COSY () and selected HMBC correlations of 1.
Figure 2. Key NOESY correlations of 1.
170.0 20.8 174.9 33.7 17.9 18.0 169.1 21.0
169.1 21.1 174.9 33.7 17.9 18.0 170.0 20.8
169.5 21.1 175.5 33.7 18.8 17.9 170.5 21.1
169.2 20.7 169.2 20.8
169.2 20.8 174.9 34.1 19.2 18.5
165.2 130.0 129.9 128.3 132.98
165.2 129.8 129.9 128.6 133.15 170.0 21.0
164.1 151.4 125.3 137.1 123.5 153.7
164.1 151.5 125.4 137.1 123.5 153.8
164.0 151.3 125.3 137.0 123.6 153.9 169.0 20.6
Data obtained from the HMBC and HSQC spectra.
(ketone), and 15, with the last bearing a free hydroxy group, according to a HMBC cross-peak correlation between OH/C-15.
The cross-peaks of the ester carbonyls with oxymethine protons showed the positions of the ester groups. Thus, HMBC correlations of H-9 with the nicotinate carbonyl (C 164.1), H-7 with the isobutyrte carbonyl (C 174.9), and H-3 with the propionate carbonyl (C 173.0) supported the locations of the nicotinate at C-9, the isobutyrate at C-7, and the propionate at C-3, respectively. In addition, HMBC cross-peaks of H-5 and H-8 with the carbonyl of both acetates (C170.0 and 169.1, respectively) were used to de?ne C-5 and C-8 as the positions of the acetoxy groups. The parallel orientation of the ester groups at C-7 and C-9 was re?ected in the up?eld shift of the methyls from the isobutyrate moiety (H 0.86 and 0.65), caused by the aromatic ring current e?ect of the nicotinate at C-9.11 The relative con?guration of 1 was assigned from its scalar and dipolar couplings (Figure 2) as well as comparison of the NMR spectroscopic data with those of closely related compounds.12?14 Assuming a H-4R-con?guration on a biogenetic basis,15 the NOE cross-peaks of this proton (H-4/H-3 and H-4/H-2) were consistent with the -orientation of both the 3-propionate and 2-methyl moieties. The 5-H-con?guration (and consequently OAc-5R) was deduced on the basis of vicinal coupling between H-4 and H-5 (J4,5 = 0 Hz), suggesting an orthogonal (trans) relationship of these protons. The NOESY correlation between H-4 and H-7 could be rationalized in terms of the 7-position of the isobutyrate group in compound 1. The absence of dipolar coupling between H-7 and H-8 was in accordance with an OAc-8R substituent. The occurrence of a NOE interaction between H-5 (H 5.43) and H-11 (H 5.92) indicated their spatial proximity and their disposition on the same () side of the macrocyclic ring. The cross-peaks observed between the pairs H-11/H-3 and H-5/H13 de?ned the 13R-orientation of the methyl group. A large coupling constant between the H-11 ole?nic protons (J11,12 = 15.5 Hz) indicated the E-geometry of the endocyclic double bond, with H-11 located at the R-side of the diterpenoid core. The occurrence of a NOE between H-12 and one of the geminal methyl groups at C-10 (H3-18, H 1.08) was used to ?x this methyl in the R-position and the remaining geminal methyl (H319) in the -position. NOE correlations between H3-19 and H-9,
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Journal of Natural Products as well as between H-8 and H-9, indicated an R-orientation of the C-9-nicotinyloxy group. A NOE interaction of OH-15 with H-5 was used to de?ne the relative con?guration at C-15, with an OH15 substituent occurring. The absence of a NOESY cross-peak between H-4 and OH-15 supported the trans-fusion of the cyclopentane ring, which is usual in jatrophane derivatives.16,17 The co-occurring euphodendrophanes B?F (2?6) were determined as being closely related to 1 structurally. With the exception of 6, they were found to di?er from euphodendrophane A (1) only in their acylation patterns. The elucidation of the structure and relative con?guration of 2?6 was based on their NMR spectroscopic data (Tables 1 and 2) and by comparison with those of 1 and known jatrophane diterpenoids.5?10 Euphodendrophane B (2), an amorphous solid, gave a [M + H]+ ion at m/z 714.3489 (calcd 714.3484) in the HRESIMS, corresponding to a molecular formula of C38H51NO12, di?ering from that of 1 by an additional 14 amu (CH2). Its 1H and 13C NMR spectra were almost identical with those of 1. The only di?erence was the occurrence of signals typical for an additional isobutyrate ester group (instead of those for a propionate ester bonded to C-3 in 1), including resonances at H 2.64 (H-20 ), 1.27 (H3-30 ), and 1.22 (H3-40 ) and C 175.6 (C-10 ), 34.28 (C20 ), 19.8 (C-30 ), and 18.5 (C-40 ). A three-bond HMBC correlation of the isobutyrate carbonyl (C-10 ) with H-3 was also observed. The above evidence was in accordance with the structure of 2 being very similar to that of 1, di?ering from the latter in the presence of a 3-isobutyrate group instead of a 3-propionate group. Additional proof for the relative con?guration of 2 being the same as that in 1 was obtained by comparison of their NOESY spectra. Compound 3 (euphodendrophane C) was isolated as an amorphous solid with the molecular formula C39H51NO13, as established by the [M + H]+ ion at m/z 742.3438 (calcd 742.3433) in the HRESIMS. 1H and 13C NMR data of 3, fully assigned through 2D NMR experiments, closely resembled those of 1. The only di?erence was the appearance of signals of an additional acetoxy group (H 2.12 and C 20.6 and 169.0) and the absence of resonances for a hydroxy group. The molecular formula of 3 was in accordance with the proposed structural elements, as evidenced in their 1H and 13C NMR spectra. The locations of the ester groups were con?rmed through HMBC cross-peaks between the ester carbonyl carbons and the neighboring methine protons. The carbonyls of the nicotinate, propionate, and isobutyrate groups exhibited cross-peaks with H-9, H-3, and H-7, respectively, whereas two of the acetoxy carbonyls gave correlations with H-5 and H-8, thus indicating the same locations of these ester groups as in 1. The remaining acetyl group (H 2.12), without any long-range correlations with the protons from the rings, was situated at the ring junction (C-15). The acetoxy group caused paramagnetic shifts of H-1R and H-5 ( = 0.55 and 0.40 ppm, respectively), while H-4 and H-13 underwent diamagnetic shifts ( = ?0.34 and ?0.64 ppm, respectively), compared to compound 1. The relative con?guration of 3, determined on the basis of a NOESY experiment, proved to be the same as in 1. Euphodendrophane C is the 15acetylated analogue of euphodendrophane A. Euphodendrophane D (4) was obtained as an amorphous solid, with a molecular formula of C37H48O12, assigned by HRESIMS from the m/z 702.3483 [M + NH4]+ ion (calcd 702.3484). Comparison of its 1H and 13C NMR data (Tables 1 and 2) with those of compound 1 showed close similarites between these compounds, again di?ering only in their ester portions. The
Figure 3. COSY () and selected HMBC correlations of 6.
H and 13C NMR spectra of 4 indicated the presence of a benzoate, an isobutyrate, and three acetates (Table 1). The presence of a benzoate ester was apparent from 1H and 13C NMR signals at H 7.42, 7.54, and 8.01 and C 165.2, 130.0, 129.9, 128.4, and 133.0, respectively. A HMBC correlation of H-8 and the carbonyl carbon of the benzoate indicated C-8 as the position of this group. The positions of the remaining ester groups, namely, 3-isobutyrate and 5,7,9-triacetate, were also determined on the basis of the long-range correlations between the ester carbonyl carbons and the neighboring oxymethine protons. The HRESIMS of euphodendrophane E (5) showed a [M + NH4]+ ion at m/z 716.3652 (calcd 716.3641), consistent with the molecular formula C38H50O12. Both the 1H and 13C NMR spectroscopic data of 5, fully assigned through 2D NMR experiments (Tables 1 and 2, respectively), closely resembled those obtained for compound 4 and di?ered only in the signals for a propionate group at C-3 instead of isobutyrate, and an isobutyrate at C-7, instead of an acetate group. The assignment of the positions of these ester groups was based on the HMBC crosspeaks of the corresponding ester carbonyl carbons (C 173.2 and 174.9) with H-3 and H-7, respectively. The remaining HMBC correlations for the rest of the molecule were in agreement with the proposed structure. Euphodendrophane F (6) was obtained as an amorphous solid. The molecular formula was determined as C40H51NO15 by HRESIMS, showing a [M + H]+ ion at m/z 786.3346 (calcd 786.3332). The structure of 6 was deduced using 1D and 2D NMR spectroscopy, as well as comparison of the NMR spectra with those of the co-occurring jatrophanes 1?5 and related analogues.11 These data indicated a jatrophane polyol structure with seven ester groups, which were identi?ed as ?ve acetates, an isobutyrate, and a nicotinate according to their NMR data (Tables 1 and 2). They could be associated with seven oxygenated sp3 carbons: one primary, four secondary, and two tertiary. The NMR spectra also showed a keto carbonyl (C 211.5, C-14), two endocyclic double bonds (5 and 11) (trans-disubstituted 11 as in 1?5 and trisubstituted 5: H 5.14 brs, C 111.7, 125.2), an aliphatic methylene (H2-1), four methyls (one secondary: H 1.17 d, H3-20, and three tertiary (H 1.07 s, 1.24 s, 1.50 s, H3-18, -19, and -16, respectively), and one sp3 quaternary carbon (C 40.1, C-10). This, in combination with the molecular formula, corresponding to 14 double-bond equivalents, and COSY and HMBC correlations (Figure 3) indicated a bicyclic diterpene (jatrophane skeleton), di?ering from 1?5 by the endocyclic (5) double bond instead of an exocyclic (6(17)) functionality. Moreover, the occurrence of a NOESY cross-peak between H-4 (H 3.17) and H2-17 (H 5.05, 5.92) was in agreement with the E-con?guration of this double bond, the same as that observed in the previously reported jatrophane-type diterpenoids from Euphorbia helioscopia.18 Three-bond HMBC cross-peaks between H-3 (H 5.47), H-8 (H 5.38), and H2-17
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Journal of Natural Products
Figure 4. Key NOESY correlations of 6.
Figure 5. COSY () and selected HMBC correlations of 7.
with the acetate carbonyl carbons (C 170.6, 168.9, and 170.3, respectively) indicated a 3,8,17-triacetoxy substitution pattern. The HMBC correlation between H-7 (H 5.05) and the isobutyrate carbonyl (C 174.8), as well as between H-9 (H 5.18) and the nicotinate carbonyl (C 163.9), were consistent with a 7-isobutyrate, 9-nicotinate structure. The carbonyl signals of the remaining acetate groups (C 168.9 and 169.5) in 6, exhibiting no HMBC correlations, were positioned at C-2 and C-15 (C 86.4, 92.8), as 2R- and 15-substituents, according to the similarity of the chemical shifts of C(1)H2, C-2, C(3)H, and C(16)H3 (Experimental Section) with those of 2,3,5,7,15-pentaacetoxy-9-nicotinoyloxy-14-oxojatropha-6(1),11E-diene from Euphorbia peplus,11 exhibiting a 3R,15-diacetate moiety. The relative con?gurations of the remaining stereocenters were deduced from the coupling constants, a NOESY experiment (Figure 4), and comparison with literature data5,11,14 of the related compounds as well as those of 1?5. The NOEs between H-8, H-9, and H3-19 ( 1.24), with the latter assigned, according to the literature,5,14,18 as 10-methyl, indicated the usual 8R,9R-diacyloxy arrangement. The cis-arrangement of H-8 and H-9 was also supported by an observed NOE correlation between these protons. On the other hand, a very small value of the J7,8 coupling constant was in agreement with the trans-orientation of the 7- and 8-acyloxy groups,5,11 and a 7-acyloxy substituent. The 13R-methyl con?guration was also supported by the similarity of the NMR data of H-13 (H 3.58), C-13 (C 43.3), H3-20 (H 1.17), and C-20 (C 20.0) to those of the co-occurring compound 3, also bearing an OAc-15 group. Euphodendriane A (7) was obtained as an amorphous solid, and its molecular formula, C31H38O7, was found to be the same as that of a phorbol ester (Euphorbia factor Pr2) isolated from Euphorbia prolifera.19 This was deduced from the [M + Na]+ peak at m/z 545.2515 (calcd for C31H38O7Na, 545.2510) in the HRESIMS. The 1H and 13C NMR spectra (Experimental Section) showed signals typical for phorbol esters with a tigliane-type diterpene skeleton.7 The 1H NMR spectra displayed signals for ?ve methyl groups (H 1.20 s, H-16; 1.34 s, H-17; 1.15 d, J = 6.5 Hz, H-18; 1.82 brs, H-19; 1.91 brs, H-20) and two ole?nic protons (H 7.07 brs, H-1 and 4.89 brs, H-7). Two 1H NMR doublets (H 5.70, J = 10 Hz; 0.82, J = 4.5 Hz) were attributed to H-12 and H-14, respectively. Three methine protons (H 3.14, dd, J = 6.5 and 4.5 Hz, H-4; 4.46, dd, J = 12.5 and 4.5 Hz, H-5; 3.65, m, H-10) were evident from the 1H NMR spectrum of 7. The COSY correlations revealed three independent spin systems (Figure 5), namely, at H3-19/H-1/H-10/H-4/ H-5, H3-20/H-7/H-8/H-14, and H3-18/H-11/H-12. A down?eld singlet at H 6.14 and a doublet at H 6.05, both without any correlations in the HSQC spectrum, were assigned to hydroxy groups at C-9 and C-5, respectively. The 1H and 13C NMR spectra also exhibited signals typical for a benzoate group (H 8.06 dd, 7.61 dt and 7.49 t; C 166.1, 129.7, 129.6, 128.5, and
Figure 6. Key NOESY correlations of 7.
133.1) and an isobutyrate group (H 2.58 sept, 1.19 and 1.16 d; C 179.6, 34.2, 18.5, and 18.5). The position of the benzoate ester at C-12 was established by a three-bond HMBC correlation between H-12 (H 5.70) and the benzoate carbonyl carbon (C 166.1). The absence of any HMBC correlations of the carbonyl (C 179.6) of the remaining ester function, assigned as an isobutyrate, indicated its attachment to a carbon bearing no hydrogen (C-13). The 13-isobutyrate ester unit was also supported by the almost identical chemical shift of C-3 (C 64.7) with that of the related 4-deoxyphorbol derivatives.8 The proposed skeletal structure of 7 was further supported by the correlations observed in the HMBC spectrum (Figure 5). The relative con?guration of 7 was deduced from the NOESY spectrum (Figure 6). All tigliane diterpenoids discovered to date possess H-8, OH-9R, and H-10R con?gurations.20 The observed NOE correlations between H-11/H-8 and H-11/H-17 indicated that these protons are on the same () side of the molecule. The coupling constant, J11,12 = 10 Hz, suggested the opposite con?guration of H-11 and H-12, and the NOE e?ect between H-12/H3-18 indicated the R-orientation of H-12. The absence of any NOE e?ect between H-8 and H-14 suggested that H-14 is R-oriented. The NOE between H3-18 and H-10 con?rmed the R-orientation of H-10. Since H-4 exhibited a NOE with H-10, both protons were assigned as R-oriented. The R-arrangement of H-4 was also supported by the absence of a NOE with H-8. Compound 7 exhibited the same gross structure as Euphorbia factor Pr2 from E. prolifera.19 According to the di?erences in 1H NMR data, most particularly those concerning H-1, H-5, H-7, and H-10, tigliane 7 was assigned as the 4-epi derivative of Euphorbia factor Pr2. Compounds 1?7 were evaluated for their capacity to inhibit the in vitro growth of four human cancer cell lines (NCI-H460, NCI-H460/R, DLD-1, and U-87 MG) using a sulforhodamine B (SRB) assay. The most e?ective were 2 and 7 (Table 3). The MDR phenotype in a NCI-H460/R cell line that was highly resistant to paclitaxel and doxorubicin did not signi?cantly change the inhibitory pattern of the compounds tested compared to the corresponding sensitive cell line, NCI-H460. Only the sensitivity to 6 was decreased considerably using the NCI-H460/ R cell line. The degree of growth inhibition of 1 and 2 did not
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Journal of Natural Products Table 3. Growth Inhibitory E?ects of Compounds Tested against Four Di?erent Cancer Cell Lines
NCI-H460 compound 1 2 4 5 6 7 paclitaxelc
NCI-H460/Rb IC30 (M) 26.6 3.9 14.6 15.2 51.9 15.7 440 IC50 (M) 44.8 17.2 30.1 49.8 90.8 26.0 2078 IC30 (M) 7.3 4.8 14.2 13.7 38.8 13.3 23
DLD-1 IC50 (M) 59.3 22.1 42.7 37.9 75.3 27.4 39 IC30 (M) 74.7 6.8 10.4 14.1 27.3 33.2 49
U-87 MG IC50 (M) 155.3 107.7 52.6 73.1 65.5 60.1 90
IC30 (M)a 31.0 6.1 11.7 8.3 4.8 7.0 3.6
IC50 (M)a 46.6 16.2 21.3 21.9 14.8 14.3 9.4
Values represent the average from ?ve independent experiments. IC30 and IC50 were calculated by linear regression analysis using Excel software. b NCI-H460/R is a multi-drug-resistant cancer cell line obtained by drug selection from its sensitive counterpart NCI-H460.4 c Data for the positive control (paclitaxel) are expressed in nM.
Table 4. Relative Reversion of Resistance to Paclitaxel and Doxorubicin by Simultaneous Treatment with Compounds 1 and 2 and Verapamil
test compound(s) paclitaxel 1 + paclitaxel 2.1 3.2 2 + paclitaxel 1.9 3.0 verapamil + paclitaxel 0.9 1.8 doxorubicin 1 + doxorubicin 2 + doxorubicin verapamil + doxorubicin FARa concentration (M) (0.005?5) 1 2.5 5 20 1 2.5 5 20 1 2.5 5 20 (0.05?5) 5 5 5 0.104 ( 0.002 0.094 ( 0.003 0.045 ( 0.017 4.307 ( 0.233 0.227 ( 0.011 0.154 ( 0.006 0.215 ( 0.013 19 28 20 CI < 0.5 (SS) CI < 0.5 (SS) CI < 0.5 (SS) 20 22 46 CI < 1 (S) CI < 0.5 (SS) CI < 0.5 (SS) IC50 (M)b 2.078 ( 0.385 0.692 ( 0.092 0.109 ( 0.003 0.055 ( 0.001 0.189 ( 0.019 0.083 ( 0.002 0.035 ( 0.005 3 19 38 11 25 60 CI < 0.5 (SS) CI < 1 (S)d CI < 0.5 (SS)e CI < 1 (S) CI < 1 (S) CI < 0.5 (SS) relative reversionc combination index (CI)
a The ?uorescence activity ratio was calculated on the basis of the measured ?uorescence values (FL2-H) expressed via the following equation: FAR = FL2-HMDR treated/FL2-HMDR control. b Results were obtained with a SRB assay. Values represent the average ( standard deviation from ?ve independent experiments. IC50 values were calculated by linear regression analysis using Excel software. c Relative reversion was calculated as IC50 for a cytostatic drug/IC50 for its combination with reversal agent. d S, synergism (CI < 1). e SS, strong synergism (CI < 0.5).
di?er substantially between the sensitive NCI-H460 and the resistant NCI-H460/R cell lines. However, the e?ectiveness of 2 was signi?cantly higher in comparison to 1 (Table 3). Since macrocyclic diterpenes of the jatrophane type previously isolated from E. dendroides have been investigated as P-gp pump inhibitors,5 it was considered that the new compounds obtained in this investigation may share such properties. Indeed, a marked increase in the accumulation of P-gp substrate Rho123 (assessed by ?ow cytometry) was observed in the MDR cancer cell line (NCI-H460/R) when treated with 1 and 2. The overexpression of mdr1 mRNA, which codes for P-gp, was reported in the NCIH460/R cell line previously.4 The e?ects of 1 and 2 on Rho123 accumulation in NCI-H460/R cells were compared with untreated resistant NCI-H460/R cells by the ?uorescence activity ratio (FAR, Table 4). Rho123 accumulation was about 2-fold higher in untreated NCI-H460 cells compared to NCI-H460/R cells. A signi?cantly higher accumulation of Rho123 in the NCI-H460/R
cell line was obtained with 1 and 2, compared to the e?ect of the standard P-gp inhibitor, verapamil (Table 4). Earlier ?ndings have highlighted the positive role of certain pharmacophoric elements in the activities of jatrophane diterpenoids toward P-gp,1 such as a free hydroxy group at C-5 and an acetate group at C-8, which are both present in jatrophanes 1 and 2. Next, the e?ects of 1 and 2, each in simultaneous combination with two anticancer agents (paclitaxel and doxorubicin), were investigated in an MDR cancer cell line. Resistant NCI-H460/R cells were treated for 72 h with combinations of 1, 2.5, and 5 M 1 or 2 and 0.005?5 M paclitaxel (Table 4). The ability of 1 and 2 to reverse drug resistance was compared to that of verapamil. As shown in Table 4, NCI-H460/R cells were also exposed to combinations of 5 M 1 or 2 and 0.05?5 M doxorubicin for 72 h. The e?ects of 1, 2, and verapamil on paclitaxel or doxorubicin sensitivity were assessed using a SRB assay. The IC50 value for paclitaxel decreased in combination with 1, demonstrating 3-,
dx.doi.org/10.1021/np200241c |J. Nat. Prod. XXXX, XXX, 000C000
Journal of Natural Products 19-, and 38-fold reversal. An even more pronounced e?ect was obtained for 2, exibiting 11-, 25-, and 60-fold reversal. There were no signi?cant di?erences in reversal activity at concentration levels of 2.5 and 5 M between compounds 1 and 2 and verapamil. Both jatrophane esters at 5 M decreased the IC50 value for doxorubicin signi?cantly, showing a similar reversal potential to verapamil (Table 4). The results obtained on combined treatment were subjected to computerized synergism/antagonism CalcuSyn software analysis. All of the combinations used in the course of treatment using resistant NCIH460/R cells induced synergistic (CI < 1) or strong synergistic e?ect (CI < 0.5). These results point to the potential of 1 and 2 to reverse paclitaxel and doxorubicin resistance in the MDR cancer cell line used. As shown earlier, a resistant NCI-H460/R cell line has been developed that displays cross-resistance to paclitaxel, vinblasine, doxorubicin, epirubicin, and etoposide.4 A synergistic interaction between jatrophane diterpenes and anthracyclines (epirubicin and doxorubicin) was found previously.21,22 Compounds 1 and 2 sensitized NCI-H460/R cells to paclitaxel in a concentrationdependent manner (Table 4). This is the ?rst report of a synergistic e?ect observed between jatrophanes and the taxane paclitaxel.
General Experimental Procedures. Optical rotations were determined on a Autopol IV (Rudolph Research Analytical) polarimeter equipped with a sodium lamp (589 nm) and 10 cm microcell. 1H and 13 C NMR data were measured on a Bruker Avance III 500 NMR spectrometer (500 MHz for 1H and 150 MHz for 13C NMR, in CDCl3, with TMS as internal reference). High-resolution LC/ESI TOF mass spectra were measured on a HPLC instrument (Agilent 1200 Series, Agilent Technologies) with a Zorbax Eclipse Plus C18 column (150 ? 4.6 mm i.d.; 1.8 m) and a diode-array detector (DAD) coupled with a 6210 time-of-flight LC/MS system (Agilent Technologies). HPLC was performed on an Agilent 1100 Series instrument in a gradient mode on Zorbax XDB-C18 column with a DAD. Detection of analytes was performed at 260 nm. Dry ?ash chromatography was performed on silica gel (70?230 mesh). Silica gel 60 F254 precoated aluminum sheets (0.25 mm, Merck) for TLC control and preparative TLC plates (2 and 0.5 mm Merck) for preparative puri?cation were used. Plant Material. The aerial parts of E. dendroides were collected around Petrovac (Montenegro) in April 2009. The spurge was identified by Petar Marin, and a voucher specimen (No. 030409) was deposited at the Herbarium of Botanical Garden Jevremovac, University of Belgrade, Belgrade (Serbia). Extraction and Isolation. To the dried plant material (1 kg) was added 60% aqueous ethanol (3 L), and the mixture was left for 10 days at room temperature with stirring. The extract obtained after filtration was lyophilized at 40 C to give a dried residue (105 g). This extract was mixed with hexane (350 mL) and placed in an ultrasound bath for 45 min, with the soluble portion decanted. The extraction was repeated under the same conditions, and the combined extracts were concentrated under reduced pressure. The hexane fraction (4.65 g) was subjected to dry flash chromatography on silica gel using toluene?ethyl acetate for elution. Fractions B?F were eluted with a higher percent of ethyl acetate in toluene. These fractions were purified by preparative TLC on silica gel plates, 20 cm ? 20 cm. Final purification was performed by HPLC to obtain diterpene compounds 1?7. Fraction B (0.26 g, eluted with 30% ethyl acetate in toluene) was subjected to preparative TLC on silica gel, using as developing system
hexane?acetone (7:3) (the plates were developed three times), to give subfraction B-1 (77.7 mg). Final puri?cation by HPLC a?orded pure compounds 4 (4.5 mg), 5 (4.8 mg), and 7 (5 mg). Fraction C (0.25 g, eluted with a second portion of 30% ethyl acetate in toluene) was subjected to preparative TLC on silica gel, eluted with hexane?acetone (7:3) (the plates were developed ?ve times), to give two subfractions. Subfraction C-1 (81 mg) was puri?ed by HPLC to give 1 (8 mg) and 2 (16 mg). Subfraction C-2 (63 mg), after puri?cation by HPLC, yielded an additional amount of 2 (8.6 mg). Preparative TLC of fraction D (0.16 g, eluted with 40% ethyl acetate in toluene) on silica gel, using hexane?acetone (7:3) (the plates were developed three times), a?orded two subfractions. Subfraction D-1 (50 mg), after HPLC puri?cation, a?orded 1 (17 mg) and 3 (2 mg). Subfraction D-2 (51 mg) on HPLC yielded additional amounts of 1 (16 mg) and 2 (8 mg). Fraction E (0.09 g, eluted with 50%?100% ethyl acetate in toluene) was subjected to preparative TLC on silica gel, in hexane?acetone (65:35) (the plate was developed four times), to a?ord three subfractions. Subfractions E-1 (12.4 mg) and E-2 (23 mg), after puri?cation by HPLC, both yielded compound 6 (2 and 4.4 mg, respectively). Puri?cation of subfraction E-3 (21 mg) by HPLC a?orded an additional amount of 1 (2.2 mg). Preparative TLC of fraction F (0.12 g, eluated with 10% methanol in ethyl acetate) on silica gel in hexane?acetone (65:35) (the plate was developed four times) a?orded subfraction F-1 (30 mg). This was puri?ed by HPLC to give an additional amount of compound 6 (2.5 mg). Euphodendrophane A (1): colorless, amorphous solid; [R]20D +31.0 (c 0.1, MeOH); UV (MeOH) max (log ) 227 (3.48), 264 (3.28) nm; IR (film) max 3446, 2926, 1738, 1278, 1240 cm?1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; HRESIMS m/z 700.3335 [M + H]+, calcd for (C37H49NO12 + H)+ m/z 700.3328. Euphodendrophane B (2): colorless, amorphous solid; [R]20D +41.6 (c 0.15, MeOH); UV (MeOH) max (log ) 217 (3.80), 264 (3.17) nm; IR (film) max 3446, 2972, 1737, 1278,1240 cm?1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; HRESIMS m/z 714.3489 [M + H]+, calcd for (C38H51NO12 + H) m/z 714.3484. Euphodendrophane C (3): colorless, amorphous solid; [R]20D +10.1 (c 0.06, MeOH); UV (MeOH) max (log ) 213 (4.13), 264 (3.60) nm; IR (film) max 2975, 1736, 1373, 1253, 1190 cm?1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; HRESIMS m/z 742.3438 [M + H]+, calcd for (C39H51NO13+H)+ m/z 742.3433. Euphodendrophane D (4): colorless, amorphous solid; [R]20D +26.0 (c 0.08, MeOH); UV (MeOH) max (log ) 228.3 (3.98), 273.4 (2.97) nm; IR (film) max 3478, 2929, 1740, 1273,1235 cm?1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; HRESIMS m/z 702.3483 [M + NH4]+, calcd for (C37H48O12 +NH4)+ m/z 702.3484. Euphodendrophane E (5): colorless, amorphous solid; [R]20D +28.0 (c 0.1, MeOH); UV (MeOH) max (log ) 229 (4.03) 273 (3.00) nm; IR (film) max 3446, 2924, 1738, 1271, 1234 cm?1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; HRESIMS m/z 716.3652 [M + NH4]+, calcd for (C38H50O12+NH4)+ m/z 716.3640. Euphodendrophane F (6): colorless, amorphous solid; [R]20D ?8.0 (c 0.1, MeOH); UV (MeOH) max (log ) 217 (4.00), 264 (3.41) nm; IR (film) max 3447, 2931, 1737,1231 cm?1; 1H NMR (CDCl3, 500 MHz) 5.99 (1H, d, J = 16.5 Hz, H-11), 5.92 (1H, brs, H-17b), 5.75 (1H, m, H-12), 5.47 (1H, brs, H-3), 5.38 (1H, brs, H-8), 5.18 (1H, d, J = 1.5 Hz, H-9), 5.14 (1H, brs, H-5), 5.05 (2H, brs, H-7, H-17a), 3.78 (1H, d, J = 16.5 Hz, H-1R), 3.58 (1H, m, H-13), 3.17 (1H, brs, H-4), 2.00 (1H, d, J = 16.5 Hz, H-1), 1.50 (3H, s, H-16), 1.24 (3H, brs, H-19), 1.17 (3H, d, J = 6.5 Hz, H-20), 1.07 (3H, brs, H-18); five OAc: 2.20 (2 ? 3H, s, OCOCH3-3, -15) 2.13 (2 ? 3H, s, OCOCH3-8, -17), 2.04 (3H, s, OCOCH3-2); OiBu-7: 2.10 (1H, bm, H-20 ), 0.94, 0.68 (2 ? 3H, two brd, J = 7 Hz, H-30 , 3H-40 ); ONic-9: 9.20, (1H, s, H-200 ), 8.80 (1H, brd, J = 5 Hz, H-600 ), 8.20 (1H, brd, J = 8 Hz, H-400 ), 7.38 (1H, dd, J = 8, 5 Hz, H-500 ); 13C NMR (CDCl3, 125 MHz) 211.5 (C, C-14), 137.1 (CH, C-12), 131.9 (CH, C-11), 125.2 (C, C-6), 111.7 (CH, C-5), 92.8
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Journal of Natural Products
(C, C-15), 86.4 (C, C-2), 80.0 (CH, C-9), 77.9 (CH, C-3), 71.2 (CH, C-8), 68.0 (CH, C-7), 67.76 (CH2, C-17), 49.9 (CH, C-4), 47.5 (CH2, C-1), 43.3 (CH, C-13), 40.1 (C, C-10), 26.9 (CH3, C-18), 24.1 (CH3, C-19), 20.0 (CH3, C-20), 18.8 (CH3, C-16); five OAc: 170.6, 170.3, 169.5 (3 ? C, CO-3, -15, -17), 168.9 (2 ? C, CO-2, -8), 22.2, 21.3, 21.2, 21.1, 20.6 (5 ? CH3, COCH3-15, -17, -8, -3, -2); OiBu-7: 174.8 (C, CO-7), 33.5 (CH, C-20 ), 18.0, 17.7 (2 ? CH3, C-30 , -40 ); ONic-9: 163.9 (C, CO-9), 153.8 (CH, C-600 ), 151.3 (CH, C-200 ), 137.1 (CH, C-400 ), 125.2 (C, C-300 ), 123.3 (CH, C-500 ); HRESIMS m/z 786.3346 [M + H]+, calcd for (C40H51NO15 + H) m/z 786.3332. Euphodendriane A (7): colorless, amorphous solid; [R]20D ?2.7 (c 0.1, MeOH); UV (MeOH) max (log ) 230 (3.58), 271 (2.72) nm; IR (film) max 3334, 2925, 1715, 1457, 1266 cm?1; 1H NMR (CDCl3, 500 MHz) 7.07 (1H, brs, H-1), 6.14 (1H, s, HO-9), 6.05 (1H, d, J = 12 Hz, HO-5), 5.70 (1H, d, J = 10 Hz, H-12), 4.89 (1H, brs, H-7), 4.46 (1H, dd, J = 12, 4.5 Hz, H-5), 3.65 (1H, m, H-10), 3.14 (1H, dd, J = 6.5, 4.5 Hz, H-4), 2.06 (1H, m, H-8), 1.91 (3H, brs, H-20), 1.85 (1H, m, H-11), 1.82 (3H, brs, H-19), 1.34 (3H, s, H-17), 1.20 (3H, s, H-16), 1.15 (3H, d, J = 6.5 Hz, H-18), 0.82 (1H, d, J = 4.5 Hz, H-14); OBz-12: 8.06, (2H, dd, J = 8.5, 1.5 Hz, H-20 , H-60 ), 7.61 (1H, t, J = 7.5 Hz, H-40 ). 7.49 (1H, t, J = 7.5 Hz, H-30 , H-50 ); OiBu-13: 2.58 (1H, h, J = 7 Hz, H-200 ), 1.19, 1.16 (2 ? 3H, two brd, J = 7 Hz, 3H-300 , 3H-400 ); 13C NMR (CDCl3, 125 MHz) 207.6 (C, C-3), 154.4 (C, C-1), 143.8 (C, C-2), 136.9 (C, C-6), 125.5 (C, C-7), 78.2 (C, C-9), 75.5 (CH, C-12), 70.7 (CH, C-5), 64.7 (C, C-13), 56.0 (CH, C-4), 47.8 (CH, C-10), 43.5 (CH, C-11), 40.1 (CH, C-8), 38.6 (CH, C-14), 27.2 (CH3, C-20), 23.8 (CH3, C-16), 25.73 (C, C-15), 16.6 (CH3, C-17), 11.8 (CH3, C-18), 10.5 (CH3, C-19); OBz-12: 166.1 (C, CO-12), 133.1 (CH, C-40 ), 129.7 (C, C-10 ), 129.6 (2 ? CH, C-20 , C-60 ,), 128.5 (2 ? CH, C-30 , C-50 ); OiBu-13: 179.6 (C, CO-13), 34.2 (CH, C-200 ), 18.5 (2 ? CH3, C-300 , C-400 ); HRESIMS m/z 545.2515 [M + Na]+, calcd for (C31H38O7 +Na), m/z 545.2510. Cells and Cell Culture. The NCI-H460, DLD-1, and U-87 MG cell lines were purchased from the American Type Culture Collection, Rockville, MD. NCI-H460/R cells were selected originally from NCIH460 cells and cultured in a medium containing 100 nM doxorubicin.4 All cell lines were subcultured at 72 h intervals using 0.25% trypsin/ EDTA and seeded into a fresh medium at the following densities: 8000 cells/cm2 for NCI-H460 and DLD-1 and 16 000 cells/cm2 for U-87 MG and NCI-H460/R.
Supporting Information. NMR spectra of compounds 1?7 are available free of charge via the Internet at http://pubs.acs.org.
*Tel: +381-11-333-66-59. Fax: +381-11-263-60-61. E mail: email@example.com (I.S.A.). Tel: +381-11-263-04-74. Fax: +381-11-263-60-61. E mail: firstname.lastname@example.org (V.V.T.).
These authors contributed equally to this work.
ACKNOWLEDGMENT This research was supported by Ministry of Science and Technological Development of Serbia (grant nos. 172053 and 41031). REFERENCES
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Chemosensitivity Determination with a Sulforhodamine B Assay. Cells grown in 25 cm2 tissue flasks were trypsinized, seeded into
flat-bottomed 96-well tissue culture plates, and incubated overnight. NCIH460 and DLD-1 cells were seeded at 2000 cells/well, while U-87 MG and NCI-H460/R cells were seeded at 4000 cells/well. Treatment with compounds 1, 2, and 4?7 (0.5?150 M) lasted 72 h. The effects of 1 and 2 in simultaneous treatment with classic chemotherapeutic drugs (paclitaxel and doxorubicin) were studied in the MDR cell line (NCI-H460/R). The chemosensitivity assay was performed after 72 h. The cellular proteins were stained with SRB, following a slightly modified protocol.23 Median Effect Analysis. The nature of the interaction between selected compounds and two classical chemotherapeutic drugs was analyzed using Calcusyn software, which uses the combination index method based on the multiple drug effect equation.24 Rho123 Accumulation Assay. Rho123 accumulation was analyzed by flow cytometry utilizing the ability of Rho123 to emit fluorescence. The intensity of the fluorescence was proportional to Rho123 accumulation. Studies were carried out with verapamil and 1 and 2. NCI-H460 and NCIH460/R cells were grown to 80% confluence in 75 cm2 flasks, trypsinized, and resuspended in 10 mL centrifuge tubes in a Rho123-containing medium. The cells were treated with 1 and 2 and verapamil (5 and 20 M) and incubated at 37 C in 5% CO2 for 30 min. The samples were analyzed using a FACScalibur flow-cytometer (Becton Dickinson, Oxford, U.K.). The fluorescence of Rho123 was assessed on fluorescence channel 2 (FL2-H) at 530 nm. At least of 10 000 events were assayed for each sample.