Madecassic Acid Derivatives as Potential Anticancer Agents

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Madecassic Acid Derivatives as Potential Anticancer Agents: Synthesis and Cytotoxic Evaluation Ana S. C. Valdeira,†,‡,§ Emad Darvishi,§ Girma M. Woldemichael,§,⊥ John A. Beutler,§ Kirk R. Gustafson,*,§ and Jorge A. R. Salvador*,†,‡ †

Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal § Molecular Targets Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States ⊥ Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, Maryland 21702-1201, United States

Downloaded via UNIV OF SOUTHERN INDIANA on July 25, 2019 at 14:44:50 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: A series of novel madecassic acid (1) derivatives was synthesized, and their cytotoxicity was evaluated against the NCI-60 panel of cancer cell lines. Several analogues exhibited broad-spectrum cytotoxic activities over all nine tumor types represented in the panel, with more potent antiproliferative activities observed against selected cancer cell lines, including multidrug-resistant phenotypes. Among them, compound 29 showed GI50 (50% growth inhibition) values ranging from 0.3 to 0.9 μM against 26 different tumor cell lines and selectivity for one colon (COLO 205) and two melanoma (SK-MEL-5 and UACC-257) cell lines at the TGI (total growth inhibition) level. The mode of action of 29 was predicted by CellMiner bioinformatic analysis and confirmed by biochemical and cell-based experiments to involve inhibition of the DNA replication process, particularly the initiation of replication, and disruption of mitochondrial membrane potential. The present findings suggest this novel madecassic acid derivative may have potential as an anticancer therapeutic lead for both solid and hematological tumors.

N

is known to possess important pharmacological activities, such as wound healing,23 antioxidant,24 anti-inflammatory,25 and antidiabetic26 effects. Furthermore, a recent study27 reported evidence for an apoptotic effect of 1 in the colon cancer cell line CT26. Compared to some other triterpenoid scaffolds, only a limited number of madecassic acid derivatives are known, and few of these have been investigated with respect to their cytotoxic activity.28,29 In order to search for novel antitumor compounds and provide insight into the influence of substituent modification at the C-2, C-3, and C-23 positions of 1 on cytotoxic activity, a library of madecassic acid derivatives comprising different ester substituents in the Aring and various functionalities in ring B was synthesized and

atural products and their derivatives remain a rich source of successful drug leads.1−5 A historical assessment of all FDA-approved new molecular entities (NMEs) reveals that natural products and their analogues represent over one-third of all NMEs.6 In the past decade there has been enormous interest in terpenoid natural products and their semisynthetic derivatives due to their remarkable structural diversity and wide range of pharmacological activities.7−10 Among all the terpenoids, pentacyclic triterpenoids are the most potent compounds endowed with anti-inflammatory and antitumor activities.11−18 There are hundreds of publications and patents describing the synthesis and biological properties of pentacyclic triterpenoids, which is indicative of wide interest in these compounds by academic and pharmaceutical industry research groups.19−21 Madecassic acid (1) is a naturally occurring pentacyclic triterpenoid found in the traditional medicinal plant Centella asiatica (L.) Urban.22 This metabolite © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 16, 2018

A

DOI: 10.1021/acs.jnatprod.8b00864 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Scheme 1. Synthesis of Madecassic Acid Derivatives 2−10

and increase their selectivity toward tumor cells.29−32 Taking this into account, a panel of 2-furoylate and cinnamate madecassic acid derivatives was prepared in an attempt to improve the compound cytotoxicity and selectivity profiles.

evaluated for in vitro cytotoxic activities. Previous studies have reported that the introduction of furoyl and cinnamic acid groups to the pentacyclic skeleton of triterpenes can significantly improve the cytotoxicity of these compounds B



Article


nih.gov/cellminer/)39 and subsequently validated by a series of cell-based and biochemical assays.

The impact of oxidative modification of the A-ring of 1 on its cytotoxic activity was also investigated. For this purpose, a series of novel diosphenol madecassic acid analogues was designed and synthesized. Accumulating evidence shows that incorporation of five-membered heterocyclic motifs into pentacyclic triterpenoid scaffolds can improve their cytotoxic activities and pharmacological properties significantly.19,33−37 Although cyclic enol ethers are found in a number of bioactive compounds,38 they have not been exploited biologically as oxygen-containing heterocycles in triterpene chemistry. This fact motivated us to synthesize a series of novel five-membered cyclic enol ethers from 1. Biological evaluation of the new madecassic acid analogues using the U.S. National Cancer Institute (NCI) 60 human cancer cell line screen revealed potent antiproliferative activity against several cancer cell lines, including multidrug-resistant phenotypes. Based on its potency and cell line selectivity, analogue 29 was identified as the most promising lead compound. The molecular mechanisms underlying the cytotoxic activity of this agent were predicted using the publicly accessible web tool CellMiner (https://discover.nci.

■

RESULTS AND DISCUSSION A series of 29 madecassic acid derivatives was synthesized, and their structures were fully confirmed by comprehensive spectroscopic analyses. With the exception of compounds 2− 4,29,40 11,29,40 12,29,41 and 19−23,29,40,42 which are known, the remaining compounds are new. As shown in Scheme 1, treatment of 1 with anhydrous potassium carbonate (K2CO3) and methyl iodide in dimethylformamide (DMF) afforded the methyl ester derivative 2 in 82% yield. Acetylation of 2 with acetic anhydride in the presence of 4-dimethylaminopyridine (DMAP) in tetrahydrofuran (THF) at room temperature gave the triacetylated derivative 3 in good yield (68%). The 2αacetylation of 2 was achieved by protection of the C-3 and C23 hydroxy groups as an acetonide 4, which allowed the DMAP-catalyzed acetylation of the C-2 hydroxy group to give intermediate 5 in high yield (76%). Deprotection of acetonide 5 with 1 M HCl in THF at room temperature gave the desired monoacetylated 6 in 87% yield. Characteristic changes in chemical shifts of key NMR signals in the starting material 2 C



Article


As depicted in Scheme 2, treatment of 3 with thionyl chloride and pyridine afforded the anhydro derivative 11, which was then deacetylated with potassium hydroxide (KOH) in MeOH to give 12. Reaction of 12 with dry acetone and a catalytic amount of HCl in the presence of activated molecular sieves gave rise to the acetonide derivative 13, which was oxidized with pyridinium dichromate (PDC) in CH2Cl2 to give the 2-oxo derivative 14. Deprotection of the acetonide group in 14 with concentrated HCl (37%) and THF under reflux provided the nor-diosphenol 15. The 1H NMR spectrum of 15 showed characteristic signals for four tertiary methyl groups at δH 2.00 (3H, s, H-24), 1.28 (3H, s, H-25), 1.03 (3H, s, H-26), and 0.93 (3H, s, H-27) and two secondary methyl signals at δH 0.92 (3H, m, H-30) and 0.83 (3H, d, J = 6.4 Hz, H-29). The methyl ester appeared as a sharp singlet at δH 3.63 (3H, s). The spectrum for 15 did not contain the methylene doublet signals for H-23 that were observed in 2. Instead, a low-field methyl signal at δH 2.00, assignable to CH3−CC, was observed for this compound. Furthermore, the 1H NMR spectrum of 15 displayed an additional CH signal at δH 6.30 (s, H-1) attributed to the olefinic proton of the enol. The presence of only 30 13C NMR signals confirmed the loss of the secondary carbon resonating at δC 68.4 assigned to C-23 in 2. The 2hydroxy-Δ1,4-dien-3-one subunit was established from key 1 H−13C correlations from the less shielded olefinic proton at δH 6.30 (H-1) to C-3 (δC 181.7), oxygenated C-2 (δC 144.4), C-9 (δC 45.1), and the C-25 methyl carbon (δC 21.7). HMBC correlations from the vinyl methyl protons (δH 2.00) to C-3

compared to the products 3 and 6 were utilized for structure characterization and product identification. The 1H NMR spectrum of 2α,3β,23-triacetate 3 revealed signals due to acetate methyl groups at δH 2.06, 2.03, and 1.98 ppm, and these showed strong HMBC correlations with oxymethine carbons at δC 65.4 (C-23), 75.0 (C-3), and 70.0 (C-2), respectively. The 1H NMR spectrum of 6 showed a deshielded shift of the H-2 signal (δH 5.08, dt, J = 10.7, 4.5 Hz), and correlations in the HMBC spectrum from both H-2 and an acetate methyl (δH 2.08, 3H, s) to the carbonyl carbon at δC 171.9 established an acetyl group attached to C-2. The 2α-(2-furoyl) and 2α-cinnamic ester derivatives 7 and 9 were synthesized by reaction of 4 with 2-furoyl chloride and trans-cinnamoyl chloride, respectively, in dry benzene and DMAP at 40 °C under a nitrogen atmosphere (Scheme 1). Deprotection of 7 and 9 with 1 M aqueous HCl in THF gave the desired 2-substitued 2-furoyl 8 and cinnamoyl-3β,6β,23trihydroxy 10 products in moderate to good yields. Characteristic signals in each 1H NMR spectrum allowed ready identification of these derivatives. The introduction of a furan ring in derivative 8 was confirmed by the presence of three aromatic peaks at δH 7.57 (1H, m, H-5′), 7.19 (1H, br d, J = 3.2 Hz, H-3′), and 6.51 (1H, m, H-4′). The 1H NMR spectrum of the 2α-cinnamic ester derivative 10 showed two multiplets, one between δH 7.53−7.51 (2H, m) and the other between δH 7.40−7.38 (3H, m) for the aromatic protons. The olefinic CH protons were observed as doublets (J = 16.0 Hz) at δH 7.70 (1H, OC−CHCH−) and 6.43 (1H, OC− CHCH−). D



Article


in 51% yield by oxidation with a mixture of potassium permanganate and iron(III) sulfate.43 The trihydroxy compound 23 was obtained in 65% yield by deacetylation of 22 and subsequently transformed into cyclic enol ether derivative 24 using the same procedure as described previously for the synthesis of 21. Cyclodehydration to the five-membered enol ether 25 was carried out by the acid-catalyzed rearrangement of 17 in 66% yield. In order to obtain the diosphenol 26, compound 25 was subjected to a base-rearrangement using 10% methanolic KOH, according to the same procedure described previously for the synthesis of 18 (Scheme 4). The IR spectrum of 26 was consistent with a diosphenol functionality with absorptions for a conjugated ketone (1674 cm−1) and an enol OH (3364 cm−1). A singlet at δH 6.11 in the 1H NMR spectrum assigned to the C-1 vinylic proton and 13C NMR signals for an olefinic carbon at δC 126.5 (assigned to C-1) and a carbonyl carbon at δC 198.6 supported a diosphenol group. We also investigated if cleavage of ring A between C-2 and C-3 of 25 would lead to a compound with more potent inhibitory effects on the growth of cancer cells. Reaction of 25 with hydroxylamine hydrochloride in EtOH in the presence of sodium acetate (NaOAc) afforded the corresponding hydroxyimino alcohol 27 (Scheme 4). The presence of a hydroxy group at C-3 was indicated by the presence of a broad IR absorption band in the 3248 to 3583 cm−1 region and by a methine singlet at δH 4.20 corresponding to H-3 in the 1H NMR spectrum. The 13C NMR spectrum contained a signal at δC 158.0, which was more

(δC 181.7), C-5 (δC 167.5), and C-4 (δC 126.0) confirmed the connectivity of this methyl group to C-4. For the preparation of 2,6-dioxo derivative 17, acetonide 4 was oxidized with PDC in CH2Cl2 to give 16, which was deprotected under acidic conditions to give 17 in 58% yield. The hydroxy ketone 17 was then subjected to a base-catalyzed enolization with 10% methanolic KOH under reflux for 3 h to furnish diosphenol 18 (Scheme 2). The NMR spectra of this compound closely resembled those of 15, with changes focused on signals associated with the B-ring. The most obvious differences were the absence of the H-6 methylene signals in the 1H NMR spectrum and the presence of an additional carbonyl signal at δC 205.9 ppm (assigned to C-6) in the 13C NMR spectrum of 18. Oxidation of 3 with Jones reagent in acetone afforded ketone 19, which was in turn treated with KOH in methanol to give the corresponding triol derivative 20 in 65% yield. Cyclodehydration of 20 under acidic conditions led to the formation of cyclic enol ether 21 (Scheme 3). The 1H NMR spectrum of this compound revealed the absence of a singlet at δH 2.48 (assigned to H-5 in 20) and a deshielded shift of the H-23 methylene protons (δH 4.21 and 4.10, 1H, d, J = 9.0 Hz, each). The absence of the carbonyl peak at δC 212.2 in the 13C NMR spectrum and the presence of an additional quaternary carbon peak at δC 114.1 assigned to C-5 confirmed the placement of a double bond at C-5−C-6 and the presence of a five-membered cyclic vinyl ether ring. As shown in Scheme 3, compound 3 was also converted to α,β-unsaturated ketone 22 E



Article

Table 1. Antiproliferative Activity of Select Madecassic Acid Derivatives compound madecassic acid (1) 2 3 6 8 10 12 15 17 18 21 22 24 25 26 27 28 29 30

NSC numbera 783350 787805 787221 787213 787214 785396 787216 783353 787807 783354 785612 783351 783352 787806 787219 787804 787220 787217 787218

one-dose mean growth (%)b

five-dose mean GI50 (μM)c e

99.2 −54.1 8.0 −79.2 −83.0 −4.8 −55.4 31.7 −22.4 67.8 85.8 45.8 51.5 −62.9 −58.9 −76.9 −18.1 27.2 78.2

NT 12.9 1.7 11.0 2.3 2.4 17.0 12.0 10.0 NTe NTe NTe NTe 18.2 8.9 6.6 4.9 2.3 NTe

five-dose GI50 range (μM)d

most sensitive cancer cell line

NTe 18.8 7.8 14.2 2.3 3.8 9.6 18.5 20.4 NTe NTe NTe NTe 9.9 18.7 16.9 12.7 99.7 NTe

HOP-92, NSCLCf PC-3, prostate K-562, leukemia UO-31, renal RXF 393, renal A498, renal SK-MEL-5, melanoma CCRF-CEM, leukemia RPMI-8226, leukemia CCRF-CEM, leukemia SR, leukemia PC-3, prostate MDA-MB-435, melanoma HOP-92, NSCLCf MOLT-4, leukemia 786-0, renal MOLT-4, leukemia COLO 205, colon RPMI-8226, leukemia

a

National Service Center number assigned by the Developmental Therapeutics Program, NCI to compounds tested in the NCI-60 assay. bGrowth percent at 10 μM vs negative control. cAverage GI50 value of each compound across the 60 cell lines. dDifference between the GI50 values for the most resistant cell line and the most sensitive cell line. eNot tested. fNon-small-cell lung cancer.

10, 26, 27, 28, and 29 had mean GI50 (concentration of compound that inhibits cell growth by 50%) values of

Madecassic Acid Derivatives as Potential Anticancer Agents

Recommend Documents