Ipomeolides A and B, Resin Glycosides from ... - ACS Publications

Centre for Natural Products and Traditional Knowledge, Indian Institute of Chemical Technology, Hyderabad 500007, India. ‡. Academy of Scientific an...
0 downloads 0 Views 3MB Size
Download PDF
Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

pubs.acs.org/jnp

Ipomeolides A and B, Resin Glycosides from Ipomoea pes-caprae and Combination Therapy of Ipomeolide A with Doxorubicin Madhu B. Sura,†,‡ Mangala G. Ponnapalli,*,†,‡ S. CH. V. A. Rao Annam,† and V. V. Pardhasaradhi Bobbili§ †

Centre for Natural Products and Traditional Knowledge, Indian Institute of Chemical Technology, Hyderabad 500007, India Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Technology (CSIR-IICT) Campus, Hyderabad 500007, India § Centre for Cellular and Molecular Biology, Hyderabad 500007, India Downloaded via AUBURN UNIV on April 25, 2019 at 05:07:15 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Two new resin glycosides, ipomeolides A (1) and B (2), both with an unusual nonlinear heteropentasaccharide core, along with five known compounds were isolated from the n-hexane/CHCl3 (1:1) extract of the aerial parts of Ipomoea pes-caprae. Ipomeolides A (1) and B (2) are macrolactone analogues of the rare (11R)-jalapinolic acid, and macrolactonization occurred at C-2 of the second saccharide moiety. Compounds 1 and 2 show structural variation even in the pentasaccharide core. The structures of 1 and 2 were established by a combination of spectroscopic techniques as well as chemical modifications such as acetyl and acetonide derivatives as well as hydrolysis products. The new glycosidic acid was named ipomeic acid (1c). Compounds 1, 1b, and 2b were evaluated for cytotoxicity against human tumor cell lines. Compounds 1b and 2b were not effective on epithelial cells, but affected survival of K-562, which is of hematopoietic origin. A sublethal concentration of compound 1 (4 μM) when used in combination with 1 μM doxorubicin, an anticancer agent, significantly enhanced cytotoxicity to tumor cells. Such combined synergistic potency against leukemia cells and the absence of effects on epithelial cells may be beneficial for chemotherapy with minimal side effects to treat CML (chronic myeloid leukemia) malignancies.

P

variations caused by peripheral acylation patterns and variable macrolactonization at C-2 or C-3 of the second saccharide moiety. Two new compounds were identified as ipomeolides A and B (1 and 2) from the n-hexane/CHCl3 (1:1) extract of the aerial parts of I. pes-caprae, which represent unusual variation even in the basic pentasaccharide core. Unlike resin glycosides, ipomeolides A and B, the fifth saccharide moiety is connected to C-3 of the second saccharide moiety. Their structures were deduced on the basis of spectroscopic techniques as well as chemical modifications such as the formation of acetyl and acetonide derivatives and hydrolysis products. The known compounds were identified as presqualene alcohol (3),16,17 icosyl (E)-3-(4-hydroxyphenyl)acrylate (4),18 β-sitosterol-3-Oβ-D-glucopyranoside (5),19 stigmasterol (6),20 and lupeol (7)20 by comparison with spectroscopic data of reported compounds. This is the first report of the natural occurrence of presqualene alcohol, although it is known synthetically16 as well as an intermediate in the biosynthesis of squalene.17 Doxorubicin (DOX), an anthracycline chemotherapeutic agent, has been used for the treatment of a variety of human

lants belonging to the morning glory family (Convolvulaceae) are a rich source of resin glycosides and have been a focus in biomedical research for their intriguing hybrid structures with promising biological profiles. Resin glycosides are a diverse class of amphiphilic glycolipids with a characteristic macrolactone framework composed of a hydrophobic (11S)-hydroxycarboxylic acid O-glycosidically linked to hydrophilic metabolic building blocks such as D-fucose, Dglucose, and L-rhamnose. These resin glycosides exhibit promising bioactivities such as cytotoxic,1,2 multidrug resistance (MDR) reversal,3−5 ionophoretic activity,6 novel Pglycoprotein inhibition, as well as phytogrowth controlling effects.7 As part of an ongoing research program on the exploration of the therapeutic potential of Indian mangrove flora,8−11 Ipomoea pes-caprae was collected at the Nizampatnam sea coast. Ipomoea pes-caprae (L.) R. Br. (Convolvulaceae) is an evergreen trailing vine that colonizes sand dunes along the tropical and subtropical regions of the world. Besides its ecological significance, I. pes-capre is used traditionally to treat hypertension, dysentery, arthritis, and skin infections caused by Mycobacterium tuberculosis12,13 and as a purgative.14,15 To date, hundreds of convolvulaceous resin glycosides have been reported, and the structural diversity arises from the multiple © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 28, 2018

A

DOI: 10.1021/acs.jnatprod.8b01100 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Metabolites isolated from Ipomoea pes-caprae and their derivatives.

with the molecular formula C63H110O24 with nine indices of hydrogen deficiency. This was further supported by the sodium adduct ion at m/z 1273.76 in its MALDITOF spectrum. Other significant MS ions at m/z 1068.5688 [M+ − 182 (C12H22O + H)], 1049.5551 [M+ − 201(C10H16O4 + H)], 903.4960 [M+ − H −183(C12H23O) − 163(C6H11O5)], 599.4520 [M+ − H − 475(C24H43O9) − 174(C8H14O4) − H], 417.5515 [M+ − 834(C41H70O17 + H)], 247.1173 [M+ − 1003 (C52H91O18)], and 205.6764 [M+ − H − 1044(C55H96O18)] were observed (Figure S134, Supporting Information). The IR spectrum of 1 displayed absorption bands for hydroxy (3442 cm−1) and ester (1723, 1064 cm−1) functionalities. It formed an octa-O-acetyl derivative (1a) on acetylation with pyridine in Ac2O. Its IR spectrum did not exhibit hydroxy group absorption bands indicating the presence of eight free hydroxy groups in 1. Compound 1 formed a triacetonide derivative (1b), which confirmed that 1 possessed three cis-related pairs of vicinal hydroxy groups. The 1H NMR data (Table 1) of 1 revealed the presence of six secondary methyls at δ 1.52 (d, J = 6.6 Hz), 1.62 (d, J = 6.0 Hz), 1.66 (d, J = 5.4 Hz), 1.61 (d, J = 5.4 Hz), 1.40 (d, J = 6.6 Hz), and 1.21 (d, J = 6.6 Hz); three terminal methyls at δ 0.94 (t, J = 7.2 Hz), 0.88 (t, J = 6.5 Hz), and 0.89 (t, J = 7.5 Hz); five anomeric protons at δ 4.75 (d, J = 7.8 Hz), 6.17 (br s), 5.49 (br s), 5.61 (br s), 5.94 (br s), and a

cancers for more than three decades. It is associated with adverse side effects causing cardiac, hepatic, and gastrointestinal toxicities. Combination chemotherapy is an effective strategy for clinical management of human cancers, as use of drugs in combination can reduce side effects, increase initial activity, or delay the onset of resistance. Doxorubicin is the principle agent used in many combination therapies.21,22 Herein, the isolation of two new resin glycosides along with five known compounds and the efficiency of combination treatment of ipomeolide A (1) with doxorubicin against human leukemia K-562 cells are reported.

■

RESULTS AND DISCUSSION Preliminary inspection of the 1D NMR data of compounds was complicated due to the presence of several overlapped oxymethine signals. However, this problem was resolved to some extent in acetonide derivatives compared to acetyl and aglycone derivatives. Equally intense anomeric carbon resonances were noticed when CDCl3 was employed as solvent rather than pyridine-d5. Compound 1 was obtained as a colorless, amorphous powder, [α]20D −34 (c 0.8, MeOH). The HRESIMS/MS data showed a deprotonated molecular ion at m/z 1249.7345 [M − H]− (calcd 1249.7309) in the negative ion mode, consistent B

DOI: 10.1021/acs.jnatprod.8b01100 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H NMR (600 MHz)a and 13C NMR (150 MHz)a Data of Compounds 1, 2, 1b, and 2b 1 position

b

fuc-1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ rha-1″ 2″ 3″ 4″ 5″ 6″ rha-1″′ 2″′ 3″′ 4″′ 5″′ 6″′ rha-1″″ 2″″ 3″″ 4″″ 5″″ 6″″ 7″″ 8″″ 9″″ rha-1‴″ 2‴″ 3‴″ 4‴″ 5‴″ 6‴″ 7‴″ 8‴″ 9‴″ jal-1 11 16 mba-1 2 3 4 2-Me dodeca-1 dodeca-12 ca-1 2 3 4 2′/6′ 3′/5′ 4′

1b

δH (J in Hz)

δC

4.75, d (7.8) 4.18, dd (9.6, 7.8) 4.00, br s 4.50* 3.78, d (6.6) 1.52, d (6.6)

104.6 80.5 73.5 73.3 71.2 17.7

4.70, 4.32, 4.37, 4.52, 3.96, 1.48,

d (7.8) m d (5.4) d (5.4) br s br s

99.5 73.9 79.8 80.4 68.8 19.8 99.1 74.2 70.1 80.4 68.9 19.1 104.9 72.8 73.0 73.7 71.1 18.9

1.34, 1.61, 5.45, 5.82, 4.56, 4.26, 4.39, 1.70, 6.17, 6.00, 4.87, 4.31, 4.37, 1.62, 5.90, 4.55, 4.59, 3.86, 4.27, 1.60,

s s br s br s br s br s m d (6.0) br s br s dd (1.8, 9.0) br s d (5.4) br s br s br s br s m br s br s

103.9 72.9 70.5 75.1 68.5 18.2

1.50, 1.56, 5.69, 3.93, 4.12, 5.32, 4.17, 1.33,

s s br s br s m m m d (6.6)

6.17, 6.02, 4.25, 4.61, 4.37, 1.62, 5.49, 5.97, 5.03, 4.61, 4.46, 1.66, 5.61, 4.70, 4.83, 4.10, 4.31, 1.61,

5.94, 4.70, 4.50, 5.81, 4.36, 1.40,

br s br s br s d (8.4) br s d (6.0) br s br s d (7.2) d (8.4) br s d (5.4) br s br s br s br s br s d (5.4)

br s br s br s t (9.6) m d (6.6)

2

δH (J in Hz)

δC 103.3 81.2 78.6 76.5 68.7 17.5 110.1 26.9 28.7 99.5 74.1 77.4 80.3 69.1 19.7 99.6 73.3 70.2 79.4 68.9 18.8 100.8 77.1 80.2 74.7 68.2 18.4 109.5 27.4 28.8 100.0 78.6 76.9 74.9 65.8 17.4 110.1 27.3 28.6 173.7 82.7 14.7 176.2 41.7 34.8 12.0 17.2 173.3 14.7

1.49, s 1.65, s 3.87, s 0.88, t (6.5) 2.51, 1.92, 0.94, 1.21,

sext (6.6) m t (7.2) d (6.6)

0.89, t (7.5)

173.3 82.6 14.6 176.6 41.9 34.8 12.1 17.4 173.4 14.6

3.82, m 0.89, t (6.6) 2.52, 1.84, 0.95, 1.20,

sext (6.6) m t (7.2) d (7.2)

δH (J in Hz) 4.76, 4.19, 4.12, 4.00. 3.78, 1.53,

d (7.8) m d (3.0) br s q (6.6) br s

6.19, 6.06, 4.37, 4.22,

br s br s br s m

1.65, 5.51, 5.99, 5.06, 4.24, 4.46, 1.68, 5.63,

d (6.0) br s br s m br s br s d (4.2) br s

4.32, br s 1.62, d (6.0)

5.86, br s 3.88, 5.77, 4.73, 1.52,

m t (9.6) d (6.6) br s

3.88, brs 0.88* 2.53, 1.92, 0.92, 1.22,

sext (6.6) m t (7.8) d (7.2)

0.88, t (6.0) 6.50, d (15.9) 7.76, d (15.9) 7.27, m, 2H 7.30, m, 2H 7.31, m

2b δC

δH (J in Hz)

104.6 80.6 73.5 73.3 71.2 17.7

4.73, 4.38, 4.39, 4.14, 4.72, 1.48,

br s m br s d (5.4) br s d (6.6)

99.1 73.9 79.8 80.5 68.8 19.9 99.4 74.2 70.1 80.5 68.9 19.3 104.9 72.9 72.6 74.5 71.3 19.0

1.35, 1.62, 5.45, 5.82, 3.94, 4.22, 3.98, 1.65, 6.16, 6.05, 4.86, 4.36, 4.39, 1.70, 5.73, 4.98, 4.63, 3.87, 4.26, 1.52,

s s br s br s br s br s br s br s br s br s m br s br s d (6.0) br s br s t (7.2) t (7.2) br s d (6.0)

100.7 73.7 82.7 75.1 68.4 18.3

1.57, 1.66, 5.75, 5.95, 3.82, 5.79, 4.26, 1.61,

s s br br m br br br

173.2 82.7 14.7 176.7 41.8 34.7 12.1 17.3 173.4 14.7 167.1 118.7 145.9 135.0 128.9 129.4 130.9

s s s s s

3.82, m 0.89, t 2.56, sext (6.6) 1.84 0.95, t (7.8) 1.24, d (7.2) 0.89, t (6.0) 6.50, d (15.6) 7.83, d (15.6) 7.40, m, 2H 7.34, m, 2H 7.35, m

δC 103.3 81.2 78.6 76.9 68.7 17.5 110.1 26.9 28.6 99.6 74.1 81.2 80.8 68.8 19.7 99.5 73.6 70.0 80.8 69.1 19.2 101.1 77.1 80.1 75.0 68.3 18.3 109.5 28.9 27.3 100.9 74.6 82.6 75.2 68.3 18.5

173.7 82.6 14.6 176.7 41.1 34.9 12.1 17.3 173.3 14.6 166.9 118.7 145.9 135.0 129.0 129.5 131.1

a

Data recorded in pyridine-d5. Chemical shifts (δ) are in ppm relative to TMS. The spin coupling (J) is given in parentheses (Hz). Chemical shifts marked with an asterisk (*) indicate overlapped signals. bAbbrivations: fuc = fucose; rha = rhamnose; jal = 11-hydroxyhexadecanoyl; mba = 2methylbutanoyl; dodeca = dodecanoyl; ca = cinnamoyl. C

DOI: 10.1021/acs.jnatprod.8b01100 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

key HMBC correlations were observed: H-1″ of α-Rha″ (δH 6.17) showed correlations with C-2′ of β-Fuc′ (δC 80.5), H-1″′ of α-Rha″′ (δH 5.49) with C-4″ of α-Rha″ (δC 80.4), H-1″″ of α-Rha″″ (δH 5.61) with C-4″′ of α-Rha″′ (δC 80.4), and H1‴″ of α-Rha‴″ (δH 5.94) with C-3″ of α-Rha″ (δC 73.9). In addition, the position of Mba was located at C-4‴″ of αRha‴″, and the n-dodecanoyl moiety at C-2″′ of α-Rha″′ was based on the long-range correlations between H-4‴″ of αRha‴″ (δH 5.81) with C-1 of Mba (δC 176.6) and H-2″′ of αRha″′ (δH 5.97) with C-1 of the dodecanoyl (δC 173.4) moiety, respectively. The position of lactonization at C-2″ of the rhamnosyl unit was established by the correlations between H-2″ (δH 6.02) of Rha″ and C-1 (δC 173.3) of the jalapinolic acid moiety, respectively. The NOESY spectrum of 1 provided the diagnostic NOE correlations between interglycosidic protons, H-1′/H-11, H-2′/H-1″, H-3″/H-1‴″, H-1″/H-1″′, and H-4″′/H-1″″. Thus, the structure of compound 1 was unambiguously established as (11R)-jalapinolic acid-11-O-α-Lrhamnopyranosyl-(1→4)-O-[2-O-(n-dodecanoyl)]-α-L-rhamnopyranosyl-(1→4)-O-[4-O-(2S-methylbutanoyl)-α-L-rhamnopyranosyl-(1→3)]-O-α-L-rhamnopyranosyl-(1→2)-O-β-Dfucopyranoside-(1,2″-lactone). Compound 2 was obtained as a colorless, amorphous powder. The HRESIMS data showed a proton molecular ion at m/z 1381.8009 [M + H]+ (calcd 1381.7884), consistent with the molecular formula of C72H116O25 with 15 indices of hydrogen deficiency. Its MALDITOFMS exhibited a sodium adduct ion at m/z 1403.82 [M + Na]+, indicating a molecular formula of C72H116O25Na. It further showed fragment ions at m/z 1375.75, 1347.70, 1127.74, 947.81, 789.23, 610.44, 593.47, and 413.33 (Figure S37, Supporting Information). It formed hepta-O-acetyl (2a) and diacetonide (2b) derivatives on treatment with Ac2O/pyridine and 2,2-dimethoxypropane/ PTSA (p-toluenesulfonic acid). The GC-MS analysis of the alkaline hydrolysate of 2 showed the presence of (S)-2methylbutyric acid and n-dodecanoic acid, while (E)-cinnamic acid was isolated and characterized by the 1H NMR data. The aqueous layer afforded a new ipomeic acid (1c) analogous to that generated from ipomeolide A (1). All the protons and carbons of compound 2 were assigned via the COSY (Figure 2; Table S1, Supporting Information) and HSQC data. Unlike ipomeolide A, compound 2 possesses an (E)-cinnamoyl group [a pair of trans-coupled olefinic protons at δH 6.50 (d, J = 15.9 Hz, H-2 of CA), δH 7.76 (d, J = 15.9 Hz, H-3 of CA) and (7.27, 7.30, 7.31, m, C6H5)]. The interglycosidic linkages, the locations of the Mba and n-dodecanoyl moieties, and lactonization were identical to those of ipomeolide A, which was confirmed by the HMBC correlations (Figure 2; Table S1, Supporting Information). The HMBC spectrum of compound 2 could not detect the position of the cinnamoyl group, while long-range HMBC couplings between H-2‴″ and CA-1 were noticed in its acetonide (2b) to locate the position of the cinnamoyl group at C-2‴″ (Table S1, Supporting Information). The acetonide protection reaction occurred at C-2″″ and C-3″″ and not at the C-3″″ and C-4″″ positions based on the HMBC correlations between H-2″″ and C-7″″ (Figure 2). Thus, the structure of compound 2 was elucidated as (11R)jalapinolic acid-11-O-α-L-rhamnopyranosyl-(1→4)-O-[2-O-(ndodecanoyl)]-α-L-rhamnopyranosyl-(1→4)-O-[2-O-(E-cinnamoyl)-4-O-(2S-methylbutanoyl)-α-L-rhamnopyranosyl-(1→ 3)]-O-α- L-rhamnopyranosyl-(1→2)-O-β-D -fucopyranoside(1,2″-lactone).

resonance of a long chain fatty acid. Its 13C NMR data (Table 1) exhibited 63 carbon resonances including signals ascribable to three carbonyls at δ 173.3, 173.4, and 176.6 and five anomeric carbons at δ 104.6, 99.5, 99.1, 104.9, and 103.9. The anomeric configuration of the fucosyl moiety was assigned as β based on the large coupling constant (J = 7.8 Hz) of H-1′,23 while the proton signals arising from the rhamnose residues at δH 6.17 (br s), 5.49 (br s), 5.61 (br s), and 5.94 (br s) were consistent with an α-configuration of the anomeric protons.24,25 Alkaline hydrolysis of 1 fragmented the macrocyclic lactone and liberated the fatty acids that esterified the oligosaccharide core and glycosidic acid. The fatty acids were identified by GC-MS analysis as n-dodecanoic acid and (2S)methylbutanoic acid based on comparison of its specific rotation value ([α]27D +18 (c 0.2, CHCl3)) with that of an authentic sample.5 Subsequent acid hydrolysis of glycosidic acid methyl ester liberated sugars and methyl (11R)hydroxyhexadecanoate (jalapinolic acid methyl ester). The (11R) configuration was assigned on the basis of Mosher’s method (Scheme 1).26,27 Macrolactones with an (R)Scheme 1. Hydrolysis of Ipomeolides 1 and 2 and Synthesis of Jalapinolic acid MTPA Esters

configurated jalapinolic acid moiety have not been reported thus far since the majority of the resin glycosides possess an (S)-configuration.1−5 Further, the position of the hydroxy group at C-11 in jalapinolic acid was ascertained based on crucial fragments observed at m/z 71, 101, and 201 in its EIMS data (Figure S128, Supporting Information). The sugars were identified as L-rhamnopyranose and Dfucopyranose by GC-MS analysis of the tetramethylsilane (TMS) derivatives.28 The glycosidic acid was confirmed as a new glycosidic acid termed ipomeic acid (1c). LiOH hydrolysis of 1 improved the yield of ipomeic acid (1c) compared to KOH. This was confirmed by the comparison of the 1H and 13C NMR data with those of the common gylocsidic acid simonic acid B (Table S2, Supporting Information) and compound 1. The 1H−1H COSY spectrum (Figure 2; Table S1, Supporting Information) of 1 showed spin systems H-2−H-3−H-4 corresponding to the 2-methylbutanoyl (Mba) moiety. The interglycosidic connectivities, locations of the ester substituents, and lactonization were confirmed by the HMBC experiments of not only ipomeolide A but also its acetonide derivative (1b), in which significant HMBC correlations were observed compared to the parent compound (Table S1, Supporting Information). The following D

DOI: 10.1021/acs.jnatprod.8b01100 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 2. Key HMBC and COSY correlations of compounds 1, 2, 1b, and 2b.

death in 11% of cells, addition of 4 μM ipomeolide A increases cell death to 47% (IC50 8.5 μM). The consequence of the synergistic effect of various concentrations of ipomeolide A (1) with 1 and 2 μM DOX was next examined. As shown in Figure 4B, an ipomeolide A concentration of 3 μM or more showed significant enhancement in cell death induced by 1 μM DOX and did not change beyond 4 μM and up to 10 μM. In contrast, 2 μM DOX combined with varying concentrations of ipomeolide A showed high cell death even at low concentration. Based on these experiments, the minimum concentration of DOX and ipomeolide A to be used in combination to induce death of K 562 cells was 1 μM DOX and 4 μM ipomeolide A. Further, the nature of cell death induced by cotreatment of ipomeolide A with doxorubicin was investigated. Expression of phosphatidyl serine at the cell surface is an important marker for identifying apoptotic cell death. It has been reported that Visucum album extract/doxorubicin cotreatment increases apoptotic response in K 562 cells.31 Annexin V/propidium iodide (PI)-treated cells revealed an increased population of apoptotic K562 cells upon cotreatment with doxorubicin and ipomeolide A, while treatment of each of the drugs alone did not cause significant cell death (Figure 4C). These results demonstrated that sublethal doses of doxorubicin in combination with ipomeolide A (1) are more potent in inducing apoptotic cell death in K 562 cells. This strategy will

Ipomeolide A (1), ipomeolide A triacetonide (1b), and ipomeolide B diacetonide (2b) were evaluated for their cytotoxicity against a panel of human cancer cell lines, namely, A-549, MDAMB-231, HL-60, MCF-7, and K 562, using the MTT assay. They differ in their ability to induce cell death in the cell lines tested. Ipomeolide A (1), ipomeolide A triacetonide (1b), and ipomeolide B diacetonide (2b) were not effective on A-549, MDAMB-231, HL-60, and MCF-7 (data not shown), whereas ipomeolide A (1) effected survival of K 562 cells (IC50 18.3 μM), which is of hematopoitic origin. The cytotoxic effects of ipomeolide A (1) in combination with sublethal doses of doxorubicin in the leukemia cell line K 562 were investigated. The dose-dependent side effects of doxorubicin are generally severe. Therefore, one of the major challenges in cancer chemotherapy is to reduce doses of chemotherapeutics, which can maintain their anticancer activity at optimal dose.29 Viscum album extract has been reported to have additive inhibitory interactions with anticancer drugs by enhancing their cytotoxic effects.30 An equal number of K 562 cells were seeded in 96-well plates exposed to various concentrations of DOX or ipomeolide A for 72 h. The number of surviving cells was determined and compared with untreated control. DOX showed minimal cytotoxicity at 1 μM with an LD50 value of 2 μM, and ipomeolide A was not toxic up to 10 μM and showed an LD50 value of 20 μM (Figure 4A). While 1 μM DOX induces cell E

DOI: 10.1021/acs.jnatprod.8b01100 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 3. Key NOESY correlations of compounds 1, 2, 1b, and 2b. Fractions A, B, and C were pooled together to furnish a dark green residue (45 g), which was subjected to silica gel column chromatography (CC) using gradient elution with n-hexane/acetone (10:90 to 70:30) to afford seven fractions. The first four fractions on repeated column chromatography afforded lupeol (20 mg), stigmasterol (50 mg), and n-icosyl (E)-3-(4-hydroxyphenyl)acrylate (10 mg). Purification of the seventh fraction furnished presqualene alcohol (7 mg), and fraction G (16 g) was adsorbed on silica gel (100−200 mesh, 12 g) using a gradient elution of n-hexane/acetone followed by MeOH. Fifty fractions (20 mL each) were collected and monitored by TLC (CHCl3/MeOH, 5:1), and identical fractions were pooled to give a mixture of resin glycosides (1.2 g). This mixture on repeated silica gel (100−200 mesh) CC using gradient elution of nhexane/acetone (9:1 to 6:4) afforded 16 subfractions, of which the nhexane/acetone (8:2) fraction afforded compound 2 (150 mg) and elution of n-hexane/acetone (6.5:3.5) afforded compound 1 (390 mg), while the remaining fractions were a mixture of both compounds. β-Sitosterol-3-O-β-D-glucopyranoside was isolated by crystallization of fraction F (200 mg). Ipomeolide A (1): colorless, amorphous powder; [α]20D −34 (c 0.8, MeOH); IR (KBr) νmax 3442, 2929, 2859, 1733, 1647, 1064 cm−1; 1H NMR and 13C NMR see Table 1; negative HRESIMS/MS m/z at 1249.7345 [M − H] − (cald for C 63 H 109 O 24 , 1249.7309); MALDITOFMS m/z 1273.76 [M + Na]+. Ipomeolide B (2): colorless, amorphous powder; [α]20D −27 (c 0.1, MeOH); IR (KBr) νmax 3435, 2928, 2862, 1723, 1637, 1063 cm−1; 1H NMR and 13C NMR see Table 1; MALDITOFMS m/z 1403.82 [M + Na]+.

therefore be useful to prevent side effects of DOX when used at high concentrations.

■

EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotations were measured on a JASCO P-2000 polarimeter. IR spectra were recorded on a Nicolet-740 FT-IR spectrophotometer. The NMR spectra were recorded with Bruker Avance (300, 400, and 500 MHz) and Bruker Avance II (600 MHz) for 1H and 75/100/125/150 MHz for 13C NMR spectra in CDCl3 and pyridine-d5 with TMS as an internal standard. Coupling constants are given in Hz. The HRESIMS experiment was performed on a Waters Xevo G2-XS QTOF mass spectrometer. The MALDI mass data were acquired on an AXIMA Performance MALDITOF mass spectrometer. Plant Material. The aerial parts of Ipomoea pes-caprae were collected in March 2016 from the Nizampatnam (latitude: 15°53′ N, longitude: 80°38′ E) coast of India and identified by Prof. B. Kondala Rao, Department of Marine Living Resources, Andhra University, Visakhapatnam. A voucher specimen (#IIC-MG-117) has been deposited at the herbarium of the Centre for Natural Products and Traditional Knowledge, I.I.C.T. Extraction and Isolation. The air-dried whole plant of I. pescaprae (8 kg) was powdered and extracted with n-hexane/CHCl3 (1:1) by Soxhlet extraction. Removal of the solvents in vacuo yielded the crude extract (101 g), which was subjected to silica gel (230−400 mesh) vacuum liquid chromatography (VLC) using gradient elution of n-hexane/acetone (50:50 to 100:0, v/v) and MeOH/acetone (10:90, 20:80, v/v) to yield eight fractions (A−H). F

DOI: 10.1021/acs.jnatprod.8b01100 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 4. Effect of varying concentration of ipomeolide A (1) in combination with doxorubicin on survival of K 562 cells. (A) Effect of cell death in K 562 cells treated with various concentrations of doxorubicin and ipomeolide A (1) for 72 h. (B) Effect of combination of doxorubicin 1 and 2 μM with different concentrations of ipomeolide A (*P < 0.05). (C) K 562 cells were treated as indicated for 72 h and stained to visualized PS on cell surface using labeled annexin V. Panels show images of cells captured using a fluorescence microscope. (+)-(1R,2R,3R)-Presqualene alcohol (3): colorless oil; [α]20D +41 (c 0.3, MeOH); 1H NMR (CDCl3, 400 MHz) δ 0.78−0.85 (m), 1.08 (s), 1.58 (s), 1.61 (s), 1.88−2.03 (m), 3.47 (dd), 3.73 (dd), 4.85 (m), 5.00−5.07 (m); 13C NMR (CDCl3, 100 MHz) δ 135.9, 134.0, 133.9, 130.3, 130.2, 123.5, 123.4, 123.3, 123.1, 122.2, 62.5, 38.7, 38.6, 35.6, 34.1, 28.0, 25.8, 25.6, 24.7, 24.2, 17.5, 16.7, 15.6, 15.0, 14.9. Jalapinolic acid methyl ester. Yellow oil; [α]20D −3 (c 0.3, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 3.67 (3H, s, H-OMe), 3.58 (1H, m, H-11), 2.30 (2H, t, J = 8.0, H-2), 1.62−1.26 (24H, m), 1.28 (br s), 0.89 (3H, t, J = 8.0, H-16); 13C NMR (CDCl3, 100 MHz) δ 174.3 (C-1), 71.9 (C-11), 51.4 (C-OMe), 37.5 (C-10, C-12), 34.1(C2), 31.9 (C-14), 29.7−29.1, 24.9 (C-9, C-13), 14.0 (C-16). Acetylation of Compounds 1 and 2. Compounds 1 and 2 (7 mg each) were dissolved in pyridine (0.2 mL), Ac2O (0.2 mL) was added, and the solution was left overnight at ambient temperature. After workup, the product was purified by recrystallization from nhexane to afford 1a (8 mg, 90%) and 2a (7 mg, 82%). Acetonide Protection of Compounds 1 and 2. Compounds 1 and 2 (10 mg each) were dissolved separately in CH2Cl2 (0.5 mL) and 2,2-dimethoxypropane (1.5 equiv), a catalytic amount of ptoluenesulfonic acid was added, and the solution was allowed to stir for 2 h at room temperature. After workup, the product was purified by VLC to afford 1a (10 mg, 91%) and 2a (9 mg, 85%). Ipomeoic Acid (1c). Compounds 1 and 2 (20 mg each) were dissolved in a mixture of tetrahydrofuran (THF) (2 mL) and H2O (0.6 mL). The mixture was allowed to stir for 5 min. LiOH·H2O (12.0 mmol) was added, and the mixture was stirred at room temperature for 12 h. After workup with EtOAc, the product was purified by silica gel flash CC (100% acetone to 20% CH3OH in acetone) to afford ipomeoic acid (1c) (8 mg, 50%) as a white solid. Alkaline Hydrolysis of the Resin Glycosides 1 and 2. A mixture of resin glycosides 1 and 2 (100 mg) was refluxed for 3 h in a solution of 5% KOH/H2O (5 mL). The reaction mixture was acidified

Octa-O-acetylipomeolide A (1a): colorless, amorphous powder; IR (KBr) νmax 2930, 2859, 1751, 1053 cm−1; 1H NMR see Supporting Information; MALDITOFMS m/z 1609.61 [M + Na]+. Ipomeolide A triacetonide (1b): colorless, amorphous powder; [α]20D −14 (c 0.4, MeOH); IR (KBr) νmax 3476, 2931, 2857, 1740, 1075 cm−1; 1H NMR and 13C NMR see Table 1; MALDITOFMS m/ z 1393.75 [M + Na]+. Hepta-O-acetylipomeolide B (2a): colorless, amorphous powder; IR (KBr) νmax 2930, 2859, 1751, 1053 cm−1; 1H NMR see Supporting Information; MALDITOFMS m/z 1697.75 [M + Na]+. Ipomeolide B diacetonide (2b): colorless, amorphous powder; [α]20D −19 (c 0.1, MeOH); IR (KBr) νmax 3483, 2931, 2857, 1732, 1637, 1050 cm−1 ; 1 H NMR and 13 C NMR see Table 1; MALDITOFMS m/z 1483 [M + Na]+. Ipomeic acid (1c): colorless, amorphous powder; [α]20D −310 (c 0.1, MeOH); 1H NMR (C5D5N, 300 MHz) δ 6.22 (br s, H-1″), 6.09 (br s, H-1″′) 5.94 (br s, H-1″″), 5.67 (br s, H-1‴″), 4.98 (br s, H2″″), 4.88 (br s, H-2″′), 4.84 (m, H-5″), 4.79 (m, H-5″″), 4.78 (d, 7.8, H-1′), 4.71 (br s, H-2‴″), 4.68 (br s, H-2″), 4.64 (m, H-3″), 4.61 (m, H-3‴″), 4.54 (m, H-3″′, H-3″″), 4.48 (m, H-4″′), 4.40 (m, H5″′), 4.31 (m, H-5‴″), 4.28 (m, H-4″), 4.25 (m, H-4″″), 4.16 (m, H3′, H-4‴″), 4.13 (m, H-2′), 3.95 (d, 3.0, H-4), 3.78 (br q, H-5), 2.50 (t, 6.9, H-jal-2), 1.59 (H-6″″), 1.58 (H-6″), 1.56 (H-6″′), 1.52 (H-6, H-6‴″), 0.90 (t, 6.6, H-jal-16); 13C NMR (C5D5N, 75 MHz) δ 177.5 (C-jal-1), 103.0 (C-1′), 101.9 (C-1″″), 101.9 (C-1‴″), 100.2 (C-1″), 99.9 (C-1″′), 80.9 (C-jal-11), 79.9 (C-4″), 79.9 (C-4″′), 77.1 (C-3″), 76.7 (C-2′), 75.2 (C-4′), 73.9 (C-3′), 72.4 (C-2‴″), 72.2 (C-4‴″), 72.0 (C-2″″), 71.6 (C-4″″), 71.4 (C-3″″), 71.2 (C-2″′), 70.9 (C-2″), 70.8 (C-3‴″), 70.5 (C-3″′), 69.7 (C-5′), 68.9 (C-5″″), 68.7 (C-5″′), 67.2 (C-5″), 65.8 (C-5‴″), 35.1−21.6 (C-2−C10 and C-12−C-15), 17.6 (C-6″), 17.4 (C-6″′), 17.1 (C-6″″), 16.8 (C-6′), 15.8 (C-6‴″), 13.0 (C-jal-16); HRESIMS m/z at 1001.5259 [M − H]+ (calcd for C40H81O23, 1001.5169). G

DOI: 10.1021/acs.jnatprod.8b01100 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

with dilute HCl and extracted with Et2O. The organic phase was washed with H2O, dried over anhydrous Na2SO4, and evaporated in vacuo. This residue upon crystallization from MeOH afforded readily (E)-cinnamic acid, as identified by 1H NMR (CDCl3): δH 7.80 (d, J = 16.0 Hz, 1H), 7.56 (dd, J = 2.1, 6.1 Hz, 2H), 7.56 (m, 3H), 9.70 (d, J = 16.0 Hz, 1H). The mother liquor was analyzed by GC-MS on model 5973N (MSD) with 6890N GC at 70 eV under the following conditions (HP-1 MS (30 M × 0.25 × 0.25) column; He, 1 mL/min; 160 °C, 2 min; 160−230 °C, 5 °C/min). The organic acid in the mother liquor was identified as n-dodecanoic acid by analysis of the GC-MS spectrum (tR 4.11): m/z 200 [M], 171, 157, 129, 115, 101, 85, 73, 60, and 43 and also by comparison with authentic samples. Purification of this mixture by CC over silica gel (100% n-hexane to 50% acetone in n-hexane) afforded 2-methylbutanoic acid (2 mg) and n-dodecanoic acid (3 mg). The aqueous layer was again extracted with n-butyl alcohol. Acid Hydrolysis and Sugar Analysis. The n-BuOH layer was evaporated in vacuo. The residue (70 mg from alkaline hydrolysis) was hydrolyzed with 1 N H2SO4 and then methylation with a catalytic amount of H2SO4 in MeOH and extracted with Et2O to yield methyl 11-hydroxyhexadecanoate. It was confirmed by the characteristic peaks in its 1H NMR spectrum at δH 0.89, 16-H (t, J = 8.0 Hz, 3H), 2.30, 2-H (t, J = 8.0 Hz, 3H), 3.58, 11-H (m), and 3.67, 11-OMe (s). The aqueous layer of the acid hydrolysis reaction contains the sugar constituents. This residue was dissolved in pyridine (0.1 mL), and 0.08 M L-cysteine methyl ester hydrochloride in pyridine (0.15 mL) was added. The mixture was kept at 60 °C for 1.5 h. After completion of the reaction, the resulting mixture was dried in vacuo, and the residue was trimethylsilylated with 1-trimethylsilylimidazole (0.1 mL) for 2 h. The mixture was partitioned between n-hexane and H2O (0.3 mL each), and the n-hexane extract was analyzed by GC-MS (Agilent Technologies, 5973N (MSD) with 6890N GC, 70 eV) under the following conditions (HP-1 MS column: 30 M × 0.25 × 0.25; He 1 mL/min; 50 °C; 2 min; 50−280 °C, Δ 10 °C/min). Analysis of the GC-MS spectrum confirmed the presence of D-fucose (tR7.19 min) and L-rhamnose (tR16.63 min.), which were further confirmed by comparison with authentic samples. Confirmation of the Absolute Configuration of Jalapinolic Acid Methyl Ester by Mosher’s Method. To a solution of 11hydroxyjalapinolic acid methyl ester (5 mg for each) with (S)- and (R)-MTPA-OH (4 mg each) in dichloromethane (DCM) were added N,N′-dicyclohexylcarbodiimide (DCC) (10.8 mg) and 4-dimethylaminopyridine (DMAP) (catalytic) at 0 °C, and the mixture was stirred at room temperature for 5 min. The reaction mixture was stirred at room temperature for 16 h. After workup with water the DCM layer was dried over anhydrous Na2SO4 and purified by VLC over silica gel to afford the pure (S)- and (R)-MTPA esters. The selected ΔδH values [ΔδH = δ(S) − δ(R)] δH = −0.5 Hz, H-2; δH = −0.5 Hz, −OCH3; δH = +1.0 Hz, H-12; and δH = +2.0 Hz, H-16 of 11-(S-MTPA)-hexadecanoic acid methyl ester and 11-(R-MTPA)hexadecanoic acid methyl ester facilitated the assignment of the (11R) absolute configuration (Scheme 1). Methyl 11 (S-MTPA)-hexadecanoate: 1H NMR (CDCl3, 500 MHz) δ 7.55 (m), 7.39 (m), 5.08 (m), 3.667 (s), 3.56 (s), 2.301 (t, J = 7.5), 1.621 (br s), 0.88 (t, J = 7.0). Methyl 11 (R-MTPA)-hexadecanoate: 1H NMR (CDCl3, 500 MHz) δ 7.54 (m), 7.39 (m), 5.08 (m), 3.668 (s), 3.56 (s), 2.302 (t, J = 7.5), 1.619 (br s), 0.876 (t, J = 7.0). Cell Lines and Cytotoxicity Assay. The cytotoxicity of compounds 1, 1b, and 2b on various human cancer cells was measured by the MTT assay using doxorubicin as positive control. Human cell lines K-562 (chronic myelogenous leukemia), MDA-MB231 (metastatic breast cancer), A-549 (lung adenocarcenoma), MCF7 (human breast adenocarcinoma), and HL-60 (human promyelocytic leukemia) were cultured in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s minimum essential medium (DMEM) containing 10% fetal calf serum. Cells were routinely passaged at 60− 70% confluency. The cells (approximately 5 000 cells) were seeded into 96-well plates (100 μL/well) for triplicate assays. The plates were returned to the incubator (37 °C in a humidified atmosphere

containing 5% CO2), and the cells were allowed to grow to confluence for 24 h. The following day, compounds of different concentrations were added. The liquid medium in the wells was replaced with the preprepared growth medium containing the appropriate drug formulation (100 μL of solution at the appropriate compound 1 and DOX concentrations). Cell viability was assessed after 48 h by adding 20 μL of MTT (5 mg/mL, Sigma) to each well for 4 h at 37 °C and 5% CO2 and the conversion of MTT into DMSO-soluble formazan by living cells was quantified by measuring the absorbance at 590 nm.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b01100.

■

FT-IR of compounds 1, 1a, 1b, 2, 2a, and 2b; HRESIMS/MS, HRESIMS, MALDITOFMS of compounds 1, 1a, 1b, 1c, 2, 2a, and 2b; 1H, 13C NMR spectra of compounds 1, 1b, 1c, 2, and 2b; 1H NMR of 1a and 2a; DEPT, 2D NMR (COSY, NOESY, HSQC, HMBC) spectra of compounds 1, 1b, 2, and 2b (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel/fax: +91-40-27160512. E-mail: [email protected]; [email protected]. ORCID

Mangala G. Ponnapalli: 0000-0001-7000-9455 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Dr. V. Radha, CCMB, for planning the cytotoxicity studies and help with writing the manuscript, Dr. T. V. Raju, IICT, for providing 2D NMR data and the director, Dr. S. Chandrasekhar, IICT, for his constant support and encouragement. This work was supported by the Science and Engineering Research Board, Government of India, through a grant awarded to M.G.P. [EMR/2015/002319].

■

REFERENCES

(1) Lilia, C.; Rogelio, P.-M.; Mabel, F.-S.; Nadia, J.-H.; Glenn, W. K.; Simon, G. J. Nat. Prod. 2008, 71, 1037−1045. (2) Tao, H.; Hao, X.; Liu, J.; Jian, D.; Fang, Y.; Gu, Q.; Zhu, W. J. Nat. Prod. 2008, 71, 1998−2003. (3) Yu, B.-W.; Luo, J.-G.; Wang, J.-S.; Zhang, D.-M.; Yu, S.-S.; Kong, L.-Y. J. Nat. Prod. 2011, 74, 620−628. (4) Gabriela, F.-G.; Nadia, J.-H.; Alejandro, Z.-D.; Rogelio, P.-M. J. Nat. Prod. 2012, 75, 93−97. (5) Wen-qiong, W.; Wei-bin, S.; Xiao-jing, L.; Min, H.; Li-jiang, X. J. Nat. Prod. 2014, 77, 2234−2240. (6) Kitagawa, I.; Ohashi, K.; Kawanishi, H.; Shibuya, H.; Shinkai, K.; Akedo, H. Chem. Pharm. Bull. 1989, 37, 1679−1681. (7) Rogelio, P.-M.; Rachel, M.; Ana Luisa, A.; Mahinda, W.; John, M. P.; Kinghorn, A. D.; Tricolorin, A. J. Nat. Prod. 1993, 56, 571− 582. (8) Appa Rao, A.; Madhu, A.; Madhu Babu, S.; Gowri, P.; Akella, V. S. S.; Jeelani, B. Sk. Org. Lett. 2015, 17, 2840−2843. (9) Gowri, P.; Annam, A.; Saidulu, R.; Sushma, S.; Madhu, A.; Raju, T. J. Nat. Prod. 2012, 75, 275−279. (10) Mangala Gowri, P.; Radhakrishnan, S.; Jeelani Basha, Sk; Sarma, A.; Madhusudana Rao, J. J. Nat. Prod. 2009, 72, 791−795.

H

DOI: 10.1021/acs.jnatprod.8b01100 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(11) Gowri, P.; Nalini, D.; Madhu Babu, S.; Haribabu, K.; Sarma, A.; Jeelani Basha, Sk. Nat. Prod. Res. 2017, 31, 63−69. (12) De Souza, M. M.; Madeira, A.; Berti, C.; Krogh, R.; Yunes, R.; Cechinel-Filho, V. J. Ethnopharmacol. 2000, 69, 85−90. (13) Pongprayoon, U.; Baeckström, P.; Jacobsson, U.; Lindström, M.; Bohlin, L. Planta Med. 1992, 58, 19−21. (14) Hernández-Carlos, B.; Bye, B.; Pereda-Miranda, R. J. Nat. Prod. 1999, 62, 1096−1100. (15) Noda, N.; Ono, M.; Miyahara, K.; Kawasaki, T.; Okabe, M. Tetrahedron 1987, 43, 3889−3902. (16) Daniel, H. R.; Eugene, C. Y.; Dale Poulter, C. J. Org. Chem. 1995, 60, 941−945. (17) Fugen, M.; John, P. C.; LeRoy, K.; Hans, C. J. Biol. Chem. 1974, 249, 3746−3749. (18) Wanda, K.; Jasmin, J. Planta Med. 1995, 61, 87−88. (19) Jayaprakasha, G.; Jadegoud, Y.; Nagana Gowda, G.; Bhimanagouda, S. P. J. Agric. Food Chem. 2010, 58, 180−186. (20) Atta-ur, R.; Seema, Z.; Iqbal Choudhary, M.; Nadeem Akhtar, M.; Shamsun, Nk. J. Nat. Prod. 2008, 71, 910−913. (21) Coiffier, B.; Lepage, E.; Briere, J.; Herbrecht, R.; Tilly, H.; Bouabdallah, R.; Morel, P.; Van den Neste, E.; Salles, G.; Gaulard, P. N. Engl. J. Med. 2002, 346, 235−242. (22) Bielack, S. S.; Smeland, S.; Whelan, J. S.; Marina, N.; Jovic, G.; Hook, J. M.; Krailo, M. D.; Gebhardt, M.; Papai, Z.; Meyer, J. J. Clin. Oncol. 2015, 33, 2279−2287. (23) Sang, S. M.; Lao, A. N.; Leng, Y.; Gu, Z. P.; Chen, Z. L.; Uzawa, J.; Fji-moto, Y. Tetrahedron Lett. 2000, 41, 9205−9208. (24) Agrawal, P. K. Phytochemistry 1992, 31, 3307−3330. (25) Bush, C. A. Bull. Magn. Reson. 1988, 10, 73−78. (26) Hai-Li, L.; Xiao-Yin, H.; Jia, L.; Guo-Rong, X.; Yue-Wei, G. Chirality 2012, 24, 459−462. (27) Thomas, R. H.; Christopher, S. J.; Feng, S. Nat. Protoc. 2007, 2, 2451−2458. (28) MacLeod, K.; Annemarie, W. J. Nat. Prod. 1997, 60, 467−471. (29) Marquette, C. MD.; Nabell, L. MD. Curr. Treat. Options in Oncol. 2012, 13, 263−275. (30) Siegle, I.; Fritz, P.; McClellan, M.; Gutzeit, S.; Murdter, T. E. Anticancer Res. 2001, 21, 2687−2691. (31) Srdic-Rajic, T.; Nevena, T. M.; Milena, C.; Ksenija, K.; Katarina, S.; Danijel, G.; Aleksandra, K. R.; Tamara, Z. Phytother. Res. 2016, 30, 485−495.

I

DOI: 10.1021/acs.jnatprod.8b01100 J. Nat. Prod. XXXX, XXX, XXX−XXX