Optimization by Molecular Fine Tuning of Dihydro-β-agarofuran

Article pubs.acs.org/jmc

Optimization by Molecular Fine Tuning of Dihydro-β-agarofuran Sesquiterpenoids as Reversers of P‑Glycoprotein-Mediated Multidrug Resistance Oliver Callies,†,§ María P. Sánchez-Cañete,‡,§ Francisco Gamarro,‡ Ignacio A. Jiménez,† Santiago Castanys,*,‡ and Isabel L. Bazzocchi*,† †

Instituto Universitario de Bio-Orgánica “Antonio González”, Departamento de Química Orgánica, and Instituto Canario de Investigación del Cáncer, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez 2, 38206 La Laguna, Tenerife Spain ‡ Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas (IPBLN-CSIC), Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento s/n, 18016 Armilla, Granada Spain S Supporting Information *

ABSTRACT: P-glycoprotein (P-gp) plays a crucial role in the development of multidrug resistance (MDR), a major obstacle for successful chemotherapy in cancer. Herein, we report on the development of a natural-product-based library of 81 dihydro-βagarofuran sesquiterpenes (2−82) by optimization of the lead compound 1. The compound library was evaluated for its ability to inhibit P-gp-mediated daunomycin efflux in MDR cells. Selected analogues were further analyzed for their P-gp inhibition constant, intrinsic toxicity, and potency to reverse daunomycin and vinblastine resistances. Analogues 6, 24, 28, 59, and 66 were identified as having higher potency than compound 1 and verapamil, a first-generation P-gp modulator. SAR analysis revealed the size of the aliphatic chains and presence of nitrogen atoms are important structural characteristics to modulate reversal activity. The present study highlights the potential of these analogues as modulators of P-gp mediated MDR in cancer cells.

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INTRODUCTION Cancer is one of the leading causes of death worldwide, and the number of new cases are expected to rise by 70% over the next two decades. 1 Multidrug resistance (MDR), which is characterized by cross-resistance to a broad spectrum of structurally and functionally unrelated chemotherapeutic drugs, is a pathophysiological phenomenon that limits the chemotherapeutic outcomes in cancer treatment in clinic.2 Increased drug efflux is related to the overexpression of transmembrane proteins belonging to the ATP-binding cassette (ABC) transporter family, whose physiological roles involve detoxification processes and tissue protection from xenobiotics. P-glycoprotein (P-gp) was the first identified drug efflux transporter3 and was detected in a large variety of different types of cancer.4,5 It was also found to affect bioavailability by decreasing drug uptake in the small intestine.6 Multifactorial mechanisms are involved in MDR, including drug compartmentalization in cellular vesicles, modification of drug targets, © XXXX American Chemical Society

defects in cellular pathways, or decreased drug accumulation in combination with increased drug efflux.7 P-gp overexpression is among the main factors involved and represents a promising target to reverse MDR of cancer cells, restoring their sensitivity to anticancer drugs. Over the past two decades, a number of strategies8 and chemical entities9 have been investigated in a continuing quest to reverse P-gp mediated MDR in cancer. These agents have shown remarkable promise in preclinical trials although, so far, none of the compounds reported have been approved by regulatory agencies. In fact, third-generation P-gp inhibitors such as tariquidar and zosuquidar, have reached phase III clinical trials but failed due to their toxicity and lack of pharmacological effects.10 Thus, the discovery of novel P-gp inhibitors with high activity and low toxicity still remains a Received: September 15, 2015

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DOI: 10.1021/acs.jmedchem.5b01429 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

challenge to pharmaceutical chemists.11 In this regard, recently, natural products have emerged as an innovative and promising alternative for the development of the next generation of P-gp inhibitors,12,13 sometimes referred to as “fourth-generation inhibitors”, with enhanced therapeutic efficacy and reduced toxicity compared to the first three generations of reversal agents. Among natural products, diterpenoids,14 cucurminoids,15 and flavonoids16 are the most extensively studied for their ability to inhibit P-gp and other ABC drug efflux transporters. In the search for new suitable molecules which could inhibit P-gp and appropriately modulate its transporter activity, we have previously reported on dihydro-β-agarofuran sesquiterpenes (DAS) from Celastraceae species, which have proved to be potent reversal agents against P-gp-dependent MDR mammalian cells17−20 and Leishmania.21 Thus, to discover effective reversal agents, the present study reports the design and synthesis of a natural-product-based library consisting of 81 DAS as a part of the molecular fine-tuning of lead compound 1,18 their evaluation as P-gp inhibitors, and a discussion on the structure−activity relationship.

corresponding esters 57−65 by treatment with different alcohols (Scheme 7). D. Influence of Nitrogen Atoms. Previous studies on the structural requirements of chemosensitizers have shown the importance of nitrogen atoms to enhance MDR reversal activity.26−28 On this basis, analogues 66−70 were prepared from compound 39 by nucleophilic substitution with different amines (Scheme 8). E. Influence of Hydrogen Bonding. Hydroxy groups are able to act as a hydrogen-bond donor and/or acceptor and have been reported to be relevant for inhibitor binding to Pgp.17,29,30 Therefore, the next approach was to limit this type of interaction by oxidation and chlorination of compound 1, yielding derivatives 71−73 (Scheme 9). The choice of a substituent such as chlorine was dictated by the steric and electronic effects that it can exert on the DAS skeleton, and because of its lipophilic character, which is opposite to that of hydroxy groups. F. Influence of π-Interactions. Double bonds are able to interact with P-gp by means of π-interactions.29,30 Thus, analogues 74 and 75 were prepared by hydrogenation to evaluate the annulation of this type of interaction and to introduce an additional degree of flexibility at C-8 position, starting from compounds 1 and 41 (Scheme 10). Comparison of aromatic and nonaromatic substituents prepared following the above design strategies, as well as compounds with additional double bonds, gave us further insights into the involvement of this type of interaction in the activity. G. Hybrid Compounds. During the past decade, pharmacological, biochemical, and biophysical evidence has suggested that hybrids may function as useful molecular probes for the discovery of novel therapeutic agents as well as dimeric inhibitors are able to establish high-affinity interactions with Pgp.31 Considering that DAS20 and flavonoids32 are potent and nontoxic chemosensitizers of P-gp mediated MDR in cancer, a series of homodimers (compounds 77, 81, and 82) and heterodimers (compounds 78 and 79) were designed and synthesized to reinforce their binding ability as reversal agents (Schemes 11−13). 2. Chemistry. In this study, considering the success of natural products as the starting point of novel drugs,33 a library of 81 analogues (2−82) based on the structure of the selected lead compound 118 was designed and synthesized. The starting material was obtained from the leaves of Celastrus vulcanicola by chromatographic fractionation of the CH2Cl2 soluble fraction of the ethanolic extract, and it was identified by comparison of its NMR data with those of an authentic sample.24 All analogues, except compounds 2,24 4718 and 48,20 are reported here for the first time. The structures of the new compounds were elucidated by spectrometric and spectroscopic methods, including 1H and 13C NMR spectra and 2D experiments (Supporting Information, Figure S2−S80) and those previously described by comparison of their spectroscopic data with those in the literature. Procedures for preparation of analogues and experimental data are included in the Supporting Information (Figure S82−S117). The first step in this task was to evaluate the influence that acyl moieties have on the P-gp-mediated MDR reversal activity of natural product 1. Therefore, acylation of the alcohol at C-6, using alkyl anhydrides, and alkyl or aryl chlorides yielded derivatives 2−28. Some noncommercial acid and anhydrides have been prepared from the corresponding carboxylic acid and dicyclohexylcarbodiimide. Because of the dissimilar reactivity of

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RESULTS AND DISCUSSION 1. Design Strategy. To elaborate an adequate design strategy in the search for more potent dihydro-β-agarofuran sesquiterpenes (DAS) to overcome MDR in cancer chemotherapy and analyze thoroughly the molecular characteristics involved, we considered data from QSAR models, molecular docking, and pharmacophore-based screening studies reported in the literature22,23 as well as our previous studies.17−20 These insights provided the rationale for systematic, semisynthetic modifications of lead compound 1 (1-acetoxy-9-benzoyloxy-8trans-cinnamoyloxy-4,6-dihydroxy-dihydro-β-agarofuran),18 a DAS previously identified as a potent P-gp-dependent MDR reversal agent, and the main secondary metabolite in Celastrus vulcanicola (Celastraceae).24 To carry out molecular fine-tuning of compound 1, modifications on its DAS scaffold were considered to modulate structure−function properties involved in plausible binding sites with the P-gp. Taken all together, seven rational approaches have been developed to optimize the efficacy and potency of this class of compounds as described below. A. Influence of Type of Ester Moieties. To improve our understanding of the effects of variations of the acyl chains in the selected platform, which may modify lipophilic and stereoelectronic properties, a series of analogues (compounds 2−46) were prepared by esterification of hydroxy groups at C-4 and/or C-6 (Schemes 1−5). B. cis−trans Isomerization Effects. It is well-known that the geometric isomers display distinct physical properties, chemical reactivity, and often have significant effects on the biological activity.25 Thus, the isomerization of a cis−trans double bond can offer an additional degree of diversity in drug design. Therefore, the cinnamate group of compound 1 was interconverted from a trans to cis configuration to yield compound 47 to further design a set of ester derivatives (compounds 48−52, Scheme 6). C. Influence of Anionic Character of Substituents. To better understand the role of an anionic acyl chain in the reversal activity, esters with a terminal carboxyl group at C-6 (compounds 53−56) were synthesized. Moreover, to get further insight into the impact of a free carboxyl group, in a next step, analogues 53−56 were converted into their B



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Scheme 1. Synthesis of Acyl Analogues 2−13a

a Reagents and conditions: (a) Ac2O, bromoacetyl bromide, propionyl chloride, butyric anhydride, octanoyl chloride, or cyclopropanecarbonyl chloride, Et3N or py, DMAP, CH2Cl2, rt; (b) chloroacetic anhydride, ZnCl2, CH2Cl2, rt; (c) (1) 2-methylbutyric acid, CH2Cl2, DCC, rt, (2) Et3N, DMAP, CH2Cl2, rt−95 °C. (d) pivaloyl chloride, Et3N, DMAP, CHCl3, 60 °C; (e) cyclohexanecarbonyl chloride, py, DMAP, PhMe, 60 °C; (f) (1) sorbic acid, CH2Cl2, DCC, rt, (2) py, DMAP, HOBt, CH2Cl2, rt.

Scheme 2. Synthesis of Acyl Analogues 14−28a

a

Reagents and conditions: (a) corresponding acyl chloride, Et3N or py, DMAP, CH2Cl2, rt; (b) isatoic anhydride or pirazinecarboxylic acid, 2,6lutidine, DMAP, respectively without or with DCC, PhMe, 110 °C; (c) (1) 4-(bromomethyl)benzoic acid, oxalyl chloride, DMF, PhMe, rt, (2) ZnO, Et3N, DMAP, CH2Cl2, ultrasound, rt, (3) HCl; 4: NaCl/H2O; (d) acyl chloride, DMAP, py and/or PhMe, 80 °C; (e) (1) 3-furoic acid, nicotinic acid, or trans-cinnamic acid, CH2Cl2, DCC, rt, (2) Et3N or py, DMAP, CH2Cl2 or PhMe, rt−95 °C.

Scheme 3. Synthesis of Analogues 29−31a

a

Reagents and conditions: (a) MsCl, Et3N, DMAP, PhMe/CH2Cl2 (1:2), 60 °C

acylating reagents, different reaction conditions were adopted and the corresponding reaction pathways are outlined in Schemes 1 and 2. It is worth noting that treatment of compound 1 with chloroacetic anhydride afforded, in addition to the expected ester 3, analogue 4, which was formed by the regioselective dehydration of the tertiary alcohol at C-4. Furthermore, treatment of compound 1 with methanesulfonyl chloride afforded derivatives 29 and 31 along with an unexpected derivative, 30 (Scheme 3), a tetracyclic sesquiterpenoid possessing an additional tetrahydrofuran ring between C-4 and C-6, which has not been previously reported

in this type of compounds. The cyclization sequence might occur through the formation of a carbocation at C-4, which is quenched by the hydroxy group at C-6 acting as an intramolecular nucleophile, and subsequent formation of an oxetane ring. Its structure was elucidated by mass and NMR data, including 2D NMR experiments. Thus, correlations between proton signals of the Me-15 and H-2β and H-6 and Me-14 in a ROESY experiment (Supporting Information, Figure S30) indicated a 4β,6β-orientation of the oxetane ring, confirming the proposed structure for compound 30. C



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Scheme 6. Synthesis of cis-Cinnamate Derivative 47 and Acyl Analogues 48−52a

The effect of acylation at C-4 or both C-4/C-6 could be evaluated by preparing analogues 32−37 (Scheme 4) from Scheme 4. Synthesis of Acyl Analogues 32−37a

a

Reagents and conditions: (a) UV, CH2Cl2, rt; (b) Ac2O, ZnO, ZnCl2, CH2Cl2, Ar, rt; (c) cyclohexanecarbonyl chloride, py, DMAP, ZnCl2, PhMe, 80 °C; (d) BzCl, DMAP, Et3N, CH2Cl2, Ar, 0 °C; (e) isatoic anhydride or pirazinecarboxylic acid, 2,6-lutidine, DMAP, respectively with or without DCC, PhMe, 110 °C. a

Reagents and conditions: (a) pivaloyl chloride, Et3N, DMAP, PhMe, 80 °C; (b) cyclohexanecarbonyl chloride, py, DMAP, PhMe, 80 °C; (c) chloroacetic anhydride, ZnCl2, CH2Cl2, rt; (d) bromoacetyl bromide, Et3N, DMAP, CH2Cl2, rt.

carboxy group on the acyl chain were synthesized (Scheme 7). Thus, treatment of compound 1 with alkyl anhydrides or aryl chlorides gave compounds 53−56. Subsequently, these analogues were acylated with MeOH, EtOH, or cyclohexanediol to afford derivatives 57−65. Although an excess of DMAP was used, treatment of compounds 53, 55, and 56 with DCC and EtOH and/or cyclohexanediol afforded the N,N′dicyclohexylurea derivatives 59, 63, and 65, respectively, as secondary products. The next strategy was focused on DAS analogues with nitrogen-bearing aliphatic side chains evaluating the influence of secondary or tertiary amine groups at the C-4 position. Thus, amine derivatives 66−70 were prepared from analogue 39 by nucleophilic substitution with different amines (Scheme 8) following the methodology developed by Park et al.36 The hydrogen-bonding ability of compound 1 was examined by transformation of the hydroxy groups into carbonyl group or chlorine atoms. Thus, oxidation with PCC afforded the 6-oxo derivative 71, while treatment with thionyl chloride gave the 4,6-dichloro-derivative 72, along with the dehydration product 73 (Scheme 9). The retention of configuration in compound 72 suggests that the reaction proceeds through carbocation

compound 1 as well as derivatives 38−46 (Scheme 5) using compound 2 as the starting material. The acylation process of compound 2 at the C-4 hydroxy group had to be optimized due to lack of efficacy of the reaction conditions used above. The best results were obtained using ZnO under ultrasound irradiation or ZnCl2 as a catalyst, which are modifications of the methodologies reported by Salvati-Niasary et al.34 and Singh et al.,35 respectively. It was noteworthy that compound 42 with a Δ4(14)-ene moiety was obtained by treatment of compound 2 with anhydride chloroacetic and ZnCl2. A second approach addressed the influence of the side chain geometry from a trans- to a cis-disposition of the cinnamate ester at C-8 position. The isomerization was carried out by ultraviolet light irradiation of compound 1, yielding analogue 47, which was further acylated to afford analogues 48−52 (Scheme 6). To explore the influence of acyl moieties with anionic character in the reversal activity, ester analogues at C-6 with a Scheme 5. Synthesis of Acyl Analogues 38−46a

a

Reagents and conditions: (a) Ac2O or butyric anhydride, ZnCl2, CH2Cl2, rt; (b) bromoacetyl bromide or 4-bromobutyryl chloride or 6bromohexanoyl chloride, Et3N, DMAP, ZnO, CH2Cl2, ultrasound, 0 °C; (c) cyclohexanecarbonyl chloride, DMAP, ZnCl2, py, rt; (d) BzCl, Et3N, DMAP, ZnCl2, CH2Cl2, rt; (e) 1-naphthoyl chloride, ZnO, CH2Cl2, ultrasound, rt. The side product 42 was obtained during the formation of derivatives 41, 45, and 46. D



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Scheme 7. Synthesis of Carboxylic Acid Analogues 53−65a

Scheme 10. Synthesis of Analogues 74 and 75a

a

Reagents and conditions: (a) H2, Pd/C, EtOAc, 4 bar, rt.

same strategy resulted unsuccessful. Furthermore, heterodimeric DAS-flavonoids were obtained starting from derivatives 53 and 54. These analogues were linked by aliphatic spacers of different chain lengths (n = 2, 3), and subsequent treatment with the P-gp flavonoid inhibitor chrysin37 (Scheme 12) to obtain analogues 78 and 79. Likewise, attempts to prepare hybrid DAS-DAS analogues linking two analogues of compound 2 through their C-4 substituents, using succinyl, glutaryl, terephthaloyl, or isophthaloyl moieties as spacers, were unsuccessful. However, when compound 2 was treated with adipoyl chloride, the desired hybrid 81 was obtained as well as the C-4 monoester derivative 80 (Scheme 13). These results indicate that a spacer group of at least six carbon atoms length is necessary to link two DAS units through C-4. On the basis of this observation, the C-4-bromoacetyl derivative 39 was coupled successfully by nucleophilic substitution, when piperazine was used as spacer, affording the hybrid analogue 82. 3. Biological Evaluation: Reversion of P-gp-Mediated Resistance to Daunomycin (DNM) and Vinblastine (VLB). The structural unrelated anticancer drugs DNM and VNB are well-known P-pg substrates.38 The biological activity of the selected natural DAS compound 118 and of its 81 analogues (2−82) was monitored through their ability to inhibit P-gpmediated DNM efflux and to reverse MDR in NIH-3T3MDR1 G-185 murine cells (MDR1 cells). Although analogues 1,18 2,18 47,18 and 4820 have been previously evaluated, they were included in this study to broaden the structure−activity studies. Flow cytometry analysis showed that 58 of them demonstrated higher P-gp inhibitory activity than the classical P-gp modulator verapamil (VRP) (Supporting Information, Table S118). In addition, the 30 most active compounds were able to block up to 75% of P-gp-mediated DNM transport, thus those analogues were selected for further studies. Afterward, the DAS concentration that inhibits DNM transport by 50% (Ki) was determined. The results demonstrated that all selected compounds have a potent P-gp binding affinity and were able to inhibit DNM transport at concentrations lower than 1.8 μM. Six analogues (24, 59, 57, 11, 50, and 58) showed lower Ki values than the lead compound 1, with values of 0.13, 0.19, 0.23, 0.24, 0.25, and 0.26 μM, respectively (Table 1). In the next step, the intrinsic cytotoxicity of the 30 selected DAS was tested against parenteral and MDR1 cells. DAS showed similar CC50 values (50% cytotoxic concentration) for both cell lines or lower CC50 values for MDR cells (Table 2) as previously described for other analogues,38 indicating that these compounds are not transported by P-gp. As expected, MDR cells were less susceptible to VRP than parental cells because it is a P-gp substrate. It should be pointed out that analogues 3 and 5 showed high cytotoxicity in both cell lines, which might be attributed to the presence of halogen substituents. As a

a

Reagents and conditions: (a) succinic or glutaric anhydride, DMAP, 2,6-lutidine, 100−110 °C; (b) terephthaloyl or isophthaloyl chloride, Et3N, PhMe, rt; (c) methanol or 1S,2S-trans-cyclohexanediol, DMAP, DCC, CH2Cl2, 0° → rt; (d) EtOH, Et3N, DMAP, DCC, CH2Cl2, Ar, 0° → rt. DCU: corresponding N,N′-dicyclohexylurea analogue (structure as indicated).

Scheme 8. Synthesis of Aminoester Analogues 66−70a

a

Reagents and conditions: (a) isobutylamine, diethylamine, piperidine, morpholine, or N-methylpiperazine, CH2Cl2, Et3N, rt.

Scheme 9. Synthesis of Analogues 71−73a

a

Reagents and conditions: (a) PCC, CH2Cl2, rt; (b) SOCl2, CH2Cl2, 0° → rt.

intermediates, which are captured by the chloride nucleophile from the less hindered front side. To analyze the influence of the flexibility and expanded π− electron system on the side chain, derivatives 74 and 75 were synthesized starting from compounds 1 and 42, respectively, by catalytic hydrogenation with Pd/C (Scheme 10). The next step was to prepare hybrid molecules of the type DAS-DAS or DAS-flavonoid linked through ester bonds. Homodimers were prepared by esterification of carboxy analogues (53, 55, and 56) with ethanediol. Thus, aliphatic carboxy analogue 53 gave the desired homodimeric compound 77, along with the monomeric derivative 76, and the N-acylurea analogue 59 (Scheme 11). However, attempts to obtain hybrid molecules connected by aromatic linkers (terephthaloyl or isophthaloyl, compounds 55 or 56, respectively) following the E



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Scheme 11. Synthesis of C-6/C-6 Homodimer 77a

a

Reagents and conditions: (a) 1,2-ethanediol, Et3N, DMAP, DCC, PhMe, CH2Cl2, rt.

Scheme 12. Synthesis of C-6/C-6 Heterodimers 78 and 79a

Table 1. Inhibition Constant (Ki)a Values of the Selected Analogues compd

a

1 2 3 5 6 7 8 11 14 24

Reagents and conditions: (a) chrysin, DMAP, DCC, CH2Cl2, rt.

Scheme 13. Synthesis of C-4/C-4 Homodimers 81 and 82a

Ki (μM)

compd

± ± ± ± ± ± ± ± ± ±

25 28 29 30 38 48 50 51 57 58

0.28 0.29 1.52 0.31 0.54 0.32 0.41 0.24 0.50 0.13

0.02 0.07 0.28 0.05 0.07 0.01 0.07 0.02 0.06 0.06

Ki (μM)

compd

± ± ± ± ± ± ± ± ± ±

59 60 61 66 69 71 72 74 75 76

1.74 1.21 0.60 0.50 0.52 0.61 0.25 0.42 0.23 0.26

0.65 0.15 0.11 0.04 0.08 0.11 0.02 0.15 0.04 0.03

Ki (μM) 0.19 0.46 0.61 0.63 0.31 0.63 1.34 0.47 0.40 0.50

± ± ± ± ± ± ± ± ± ±

0.03 0.09 0.13 0.11 0.02 0.17 0.21 0.07 0.11 0.18

a

Ki: concentration of compounds that inhibits 50% of P-gp-mediated DNM transport. Results are expressed as the mean ± SD of three independent experiments performed in triplicate.

Therefore, the 21 selected DAS derivatives were assayed for their ability to reverse DNM and VLB resistance in MDR1 cells (Table 3). Among them, 18 analogues exhibited strong reversal activity against DNM resistance at all concentrations (1, 3, and 10 μM) with reversal index values (RI, the ratio between the IC50s values of MDR1 cells without and with DAS) higher than the classical P-gp modulator VRP. In particular, promising reversal activities were observed for compounds 6, 24, 28, 59, and 66, which showed RI values between 3-fold and 6-fold higher than VRP at 1 μM. Furthermore, 3 μM of compound 48 and 10 μM of compounds 59 and 66 completely reverse DNM resistance. Regarding the reversal activity against VLB, two analogues (29 and 69) had RI values higher than VRP at all the assayed concentrations. Compound 59 was able to reverse VLB resistance with RI value 3-fold higher than VRP at 1 μM, although was less active at 10 μM. Furthermore, at 3 μM, compounds 6, 24, 29, 30, and 38 showed RI values between 2fold and 3-fold higher than VRP, whereas at 10 μM compounds 29 and 76 showed higher RI values than VRP, and compound 69 completely reverses VLB resistance. A graphical summary of the effectivity of the reversal activity in MDR1 cells of the selected DAS related to VRP at 1 μM (Supporting Information, Table S120) is shown in Figure 1. The overall results of the biologicals assays allowed identifying six DAS analogues (59, 28, 24, 6, and 66) with improved activity profile compared to the lead compound 1 and the reference drug VRP.

a

Reagents and conditions: (a) adipoyl chloride, Et3N, DMAP, DCC, CH2Cl2, rt; (b) bromoacetyl bromide, Et3N, DMAP, ZnO, CH2Cl2, ultrasound, 0 °C; (c) piperazine, Et3N, CH2Cl2, Ar, rt.

result, 21 compounds showing low cytotoxicity were selected for further studies. P-gp has been shown to bind structurally unrelated compounds with different binding mechanisms.39 Previous works have reported that DAS do not interact with P-gp at the anticancer drugs DNM and VLB binding sites.38,40 This efficiency in blocking DNM transport is linked to their ability to sensitize MDR1 cells to these two anticancer drugs, reverting more efficiently resistance to VLB than to DNM.38,40 F



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Table 2. Intrinsic Cytotoxicity (CC50)a of Selected DAS in Parental and MDR1 Cells compd

CC50 μM (MDR1 cells)

1 2 3 5 6 7 8 11 14 24 25 28 29 30 38 48

25.5 ± 6.9 18.3 ± 3.6 5.3 ± 0.5 5.7 ± 1.2 24.9 ± 4.2 18.3 ± 0.4 13.2 ± 2.8 11.2 ± 4.8 30.6 ± 7.7 32.6 ± 4.3 >60 (86.2 ± 6.0) 24.0 ± 7.4 31.7 ± 5.3 27.9 ± 7.4 51.6 ± 3.6 22.9 ± 2.8

CC50 μM (parental cells) 26.1 ± 7.1 26.3 ± 5.0 7.4 ± 2.1 8.6 ± 1.0 >60 (68.6 ± 44.9 ± 4.7 >60 (57.3 ± 50.3 ± 6.2 >60 (64.1 ± >60 (67.7 ± >60 (66.3 ± 56.3 ± 7.8 >60 (69.1 ± 29.4 ± 2.2 >60 (68.1 ± 12.0 ± 1.3

6.3)b 2.2) 4.5) 7.9) 5.6) 1.4) 4.2)

compd

CC50 μM (MDR1 cells)

CC50 μM (parental cells)

50 51 57 58 59 60 61 66 69 71 72 74 75 76 VRP

25.9 ± 5.1 31.2 ± 5.4 17.6 ± 5.2 15.3 ± 8.2 >60 (72.8 ± 5.2) 19.9 ± 8.8 28.2 ± 6.3 34.6 ± 4.5 33.1 ± 6.5 34.8 ± 4.8 31.4 ± 5.3 30.3 ± 6.0 14.2 ± 2.7 29.2 ± 7.8 >60 (76.9 ± 7.3)

27.2 ± 3.7 >60 (68.3 ± 5.9) 36.4 ± 4.1 58.9 ± 3.9 56.4 ± 7.2 24.3 ± 3.4 >60 (71.3 ± 6.8) 37.3 ± 5.6 36.2 ± 8.7 18.9 ± 4.1 43.7 ± 3.2 25.4 ± 1.9 >60 (72.3 ± 3.4) 27.7 ± 0.2 27.0 ± 4.1

CC50 values (μM) of DAS were determined as described in the Biological Studies section. Results are expressed as the mean ± SD of three independent experiments performed in duplicate. Concentrations above 60 μM were not tested due to the solvent (DMSO) toxicity. Compd: compounds. VRP: verapamil. bIn bracket, % of cell growth at 60 μM with respect to the control without compounds. a

Table 3. Drug Resistance Reversal Activity of DAS in MDR1 Cellsa RI with DNMb ± SD compd 1 6 14 24 25 28 29 30 38 48 50 51 59 60 61 66 69 71 72 74 76 VRPd

1 μM 5.6 10.8 6.2 11.8 2.3 11.8 7.0 5.9 6.9 7.0 4.1 3.2 15.8 3.90 6.0 10.0 5.7 5.2 5.2 3.3 2.6 2.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 3.5 1.1 2.1 0.2 5.2 1.6 3.4 3.2 1.8 1.1 0.9 4.2 0.7 1.1 2.1 1.4 1.5 0.3 0.2 0.2 0.2

3 μM 9.1 11.5 10.1 14.7 4.0 13.9 11.1 8.7 10.3 24.0 14.5 5.2 19.5 11.0 12.3 15.1 10.9 10.4 12.9 8.1 7.5 6.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 1.7 2.3 1.3 0.9 3.2 1.6 2.4 1.6 6.4 2.9 2.2 3.0 2.8 2.7 0.4 3.4 0.7 1.3 2.8 1.9 2.4

RI with VLBc ± SD 10 μM 15.1 12.4 17.5 16.1 9.0 16.3 16.3 15.6 15.2 30.3 14.7 9.0 24.9 12.8 15.9 22.2 16.1 15.9 14.8 11.6 10.6 10.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.7 0.5 4.0 0.8 0.6 2.8 0.2 1.2 3.3 6.5 4.3 2.5 2.0 2.1 3.2 2.8 1.7 2.8 5.1 3.0 1.8 2.1

1 μM 23.7 19.1 13.9 17.4 3.7 11.2 18.5 15.2 21.2 12.1 15.5 5.2 27.4 9.9 7.5 20.6 21.1 11.6 11.6 11.1 6.5 8.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.1 3.6 1.6 7.1 0.3 0.9 3.2 1.5 4.4 1.9 1.9 1.3 2.6 1.3 2.0 1.6 0.9 1.5 3.2 2.2 0.9 1.1

3 μM 30.3 69.0 33.6 56.0 8.8 37.7 86.6 75.8 58.2 19.7 30.1 18.7 36.1 40.3 37.7 49.1 49.6 29.4 33.0 23.6 26.8 27.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.5 3.3 7.2 0.6 1.6 6.4 7.2 3.1 1.2 3.1 5.9 2.5 3.5 4.3 6.9 7.1 0.8 7.5 11.9 2.4 1.7 2.6

10 μM 86.6 110.9 126.2 113.5 20.6 90.2 149.2 125.2 91.7 77.0 59.5 39.6 90.0 107.8 103.6 82.9 173.4 106.3 113.9 43.8 145.9 129.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

8.5 6.4 8.7 9.7 1.3 9.3 8.8 8.9 9.8 8.8 4.8 4.4 7.5 7.6 8.5 5.5 9.4 6.8 9.2 6.1 1.6 4.2

a

The reversal index (RI) was defined as ratio between IC50 values of MDR1 cells without and with DAS. IC50 values were determined as described in the Biological Studies section. Results are expressed as the mean ± SD of two independent experiments performed in triplicate. bThe maximum RI (ratio between IC50 for MDR1 and parental drug-sensitive cells) with DNM was 22. cThe maximum RI with VLB was 152. dVRP: verapamil.

4. Structure−Activity Relationship Analysis. To identify more thoroughly the molecular characteristics involved in P-gp inhibition and reversion of MDR in cancer cells, a structure− activity relationship (SAR) study was carried out taking into consideration Ki values for DNM transport (Table 1), MDR reversal indices (RI, Table 3), and inhibition percentages of DNM transport (Supporting Information, Figure S118). This study revealed the following trends. A. Influence of Type of Ester Moieties. Analysis of the Ki values of analogues 2, 3, 5−8, and 11 indicated no clear relation

We have reported that DAS reverse P-gp-mediated MDR because they function as inhibitors of P-gp drug transport activity.38,40 It is well-known that good P-gp inhibitors are usually poor substrates. DAS are poorly transported by P-gp, which is consistent with the inability of P-gp to confer resistance to these compounds, as shown in this work. Moreover, it has been described that DAS have long half-life of intracellular activity, which support the potential of DAS analogues reported herein to efficiently inhibit P-gp. G



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identification of compound 59 (RIDNM 15.8, 19.5, and 22.2 at 1, 3, and 10 μM) as the most active analogue of the series. D. Influence of Nitrogen Atoms. Among analogues with nitrogen atom-bearing C-4 substituents (compounds 66−70), those with an isobutylamine (66) or a morpholine (69) moiety showed Ki values of 0.63 and 0.31 μM, respectively. Moreover, compound 69 was able to completely revert VLB resistance at 10 μM, and compound 66 was nearly twice as effective as compound 1. Thus, the presence of nitrogen atoms seems to enhance the activity as demonstrated by compounds 24, 28, and 59. E. Influence of Hydrogen Bonding. Oxidation (analogue 71, Ki 0.63 μM) and esterification (e.g., analogue 2, Ki 0.29 μM) indicated that H-bond donors at C-6 position do not seem to have much impact on the activity, making this position appropriate to introduce additional substituents. However, when comparing compounds 1, 2, 3, and 29 (83−87% of DNM retention) with their corresponding acetylated (38, 85% DNM retention) and dehydrated products (73, 4, and 31), the P-gp inhibition decreased (69−66% DNM retention). This indicated that H-bond donors at C-4 position are more important than H-bond acceptors, although both are involved in the activity. F. Influence of π-Interactions. Additional π-interactions at C-4 and C-6 positions were unfavorable for compounds without nitrogen atoms (4, 13−23, 26−27, and 42). Moreover, the C-8 cinnamoyl double bond does not play a significant role, as the corresponding hydrogenation slightly decreased P-gp inhibition [Ki (1) 0.28 μM vs Ki (74) 0.47 μM] and reversion of DNM and VLB resistance. G. Hybrid Compounds. As a general trend, hetero(compounds 78 and 79) and homodimers (81 and 82) showed a moderate to significant loss of inhibitory activity depending on the type of linker, which seems to affect significantly the binding affinity to P-gp. This was evident in DAS-crysin hybrids, as analogue 78 with a succinyl linker was 5fold more potent than analogue 79 with a glutaryl linker. The aforementioned SAR study of DAS analogues provided insightful information regarding the structural features required for resistance reversion, which could be exploited in the future design of P-gp inhibitors. The side chain length and the introduction of nitrogen on the ester moiety led to the development of potent derivatives exhibiting submicromolar activities, as shown in the most active analogues 6, 24, 28, 59, and 66.

Figure 1. MDR reversal activity for DNM (green shades) and VLB (blue shades) related to VRP of the 21 selected DAS compounds at the concentration of 1 μM in MDR1 cells. Reversal index factor: RI compound/RI verapamil (VRP). Area surrounded by dotted lines includes DAS compounds that are 3-fold more effective than VRP.

between the aliphatic acyl chain at C-6 and their P-gp inhibitory activity. However, compound 6 with three carbon atoms was the less toxic or, in other words, the most specific, and showed greater reversion of DNM resistance than the lead compound 1 (RIDNM 10.8 and 5.6 at 1 μM, respectively). By contrast, analogues with an aromatic C-6 acyl moiety led to either a slight (compound 14) or drastic (compounds 15−23, 25−27) decrease in activity. An exception is compound 24, with a C-6 nicotinoyl group that showed the lowest Ki value (0.13 μM) of all the assayed DAS and at 1 μM exhibited over 2-fold and 4fold higher reversion of DNM resistance than compound 1 and VRP, respectively. This revealed the C-6 nicotinoyl group as a key structural requirement in the MDR reversal activity. Moreover, although carboxylic acid analogues (compounds 28 and 29) showed higher Ki values (1.21 and 0.60 μM, respectively) than compound 1 (0.28 μM), both compounds were more effective in reverting DNM resistance at 1 μM (RI 11.8, and 7.0, respectively). This discrepancy could be due to other factors such as increased intracellular retention of compounds or generation of more active metabolites. Furthermore, analysis of the acylated analogues at C-4/C-6 (34−37) or C-4 (32, 33, and 39−46) suggest that steric hindrance at the C-4 position is an unfavorable factor for the activity. B. cis−trans Isomerization Effects. No clear conclusion can be drawn for the effect of cis−trans isomerization, as some trans-compounds showed lower Ki values than their corresponding cis-analogue (0.29 vs 0.61 μM, respectively, for compounds 2 and 48), whereas others showed higher Ki values [Ki (14) 0.50 μM vs Ki (50) 0.25 μM, and Ki (25) 1.74 μM vs Ki (51) 0.42 μM]. A similar trend was observed for the corresponding RI values. These observations suggest a cooperative interaction of both C-6 and C-8 substituents, which seems to be involved in the reversal activity. C. Influence of Anionic Character of Substituents. Analogues with a terminal carboxyl group (derivatives 53− 56) displayed low inhibition, whereas esterification thereof (compounds 57−65) led to a complete or partial restoration of the activity. In this regard, the best fitting was obtained by the succinyl moiety, as shown by compounds 57 (OMe), 58 (OEt), and 59 (dicyclohexylurea) with similar or higher P-gp inhibition (Ki 0.23, 0.26, and 0.19 μM, respectively) than compound 1. Furthermore, their RI values permitted the

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CONCLUSIONS In summary, by carrying out a molecular fine-tuning on the dihydro-β-agarofuran skeleton, we were able to design a series of more potent reversers of P-gp-mediated MDR. During the development of our compound library, different issues, such as acyl chains, cis/trans-isomerization effects, anionic substituents, presence of nitrogen atoms, hydrogen bonding, π-interactions, or hybrid compounds were taken into consideration. The chemomodulation results of this series of DAS analogues led to the discovery of five analogues (6, 24, 28, 59, and 66) with an improved activity profile compared to the lead compound 1 and to the reference P-gp modulator VRP. The combined results of SAR analysis of these P-gp inhibitors expand our understanding of the involved optimal pharmacophore. These findings support the notion that this type of sesquiterpenoids could be considered as active inhibitors of P-gp function and shed light on the design of new MDR-modulators. H



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Article

absence of different concentrations of each DAS. All intracellular fluorescence values were converted to percentage inhibition of P-gp normalized for MDR1 cells treated with 5 mM sodium orthovanadate (100% inhibition of P-gp) and plotted and fitted to the equation I = (Imax × S)/(Ki + S) using SigmaPlot 2000 software, where I is the percentage inhibition of DNM efflux at a given DAS concentration, Imax is the maximal percentage inhibition, and S is the concentration of DAS. Reversion of P-gp-Dependent DNM and VLB Resistance. The dose−response curves of drug-sensitive and MDR1 cells were determined by the MTT colorimetric assay, 41 as described previously.19 To assess the chemosensitizing effect of the best DAS blocking the DNM efflux activity of P-gp, both drug-sensitive and MDR1 cells were exposed to increasing concentrations of DNM or VLB (up to 500 ng/mL) in the presence or absence of fixed concentrations (1, 3, and 10 μM) of each DAS. Dose−response curves were generated by nonlinear regression (using SigmaPlot 2000 for Windows, SPSS, Inc.) of the data points to a four-parameter logistic curve in order to determine CC50 (50% cytotoxic concentration) for DAS and IC50 (defined as the drug concentration that inhibits cell growth by 50%) for DNM and VLB at each DAS concentration. The reversal index for a given DAS at a given concentration is the ratio between the IC50 for MDR cells without DAS and the IC50 with DAS.

EXPERIMENTAL SECTION

General Methods for Chemistry. Optical rotations were measured on a PerkinElmer 241 automatic polarimeter in CHCl3 at 20 °C. UV spectra were obtained on a JASCO V-560 spectrophotometer in absolute EtOH. IR (film) spectra were measured on a Bruker IFS 55 spectrophotometer. 1H (400, 500, or 600 MHz) and 13 C (100, 125, or 150 MHz) NMR spectra were recorded on a Bruker Avance 400, 500, and 600 spectrometers; chemical shifts were referred to the residual solvent signal (CDCl3: δH 7.26, δC 77.0); DEPT, COSY, ROESY (spin lock field 2500 Hz), NOESY (mixing time 500 ms), HSQC, and HMBC (optimized for J = 7.7 Hz) experiments were carried out with the pulse sequences given by Bruker. MS-EI and HRMS-EI were obtained on a Micromass Autospec spectrometer, and HRMS-ESI (positive mode) was measured on a LCT Premier XE Micromass electrospray spectrometer. Silica gel 60 (particle size 15− 40 and 63−200 μm, Machery-Nagel) and Sephadex LH-20 (Pharmacia Biotech) were used for column chromatography, while silica gel 60 F254 (Machery-Nagel) was used for analytical and preparative TLC. The zones were visualized by UV light and heating the silica gel plates sprayed with H2O−H2SO4−HOAc (1:4:20). Centrifugal preparative TLC (CPTLC) was performed using a Chromatotron (Harrison Research, Inc., model 7924T) on 1, 2, or 4 mm silica gel 60 PF254 disks. Varian high-performance liquid chromatography (HPLC) equipment consisted of a ProStar 210 solvent delivery module, ProStar 335 photodiode array detector, using a semipreparative AscetisSi column (25 cm × 21.2 mm, 10 μm) and an analytical AscetisSi column (25 cm × 4.6 mm, 5 μm) with a flow rate of 0.5 mL/ min). The degree of purity of the new compounds was over 95%, as indicated by the appearance of a single peak using HPLC. All solvents used were analytical grade (Panreac). The reagents were purchased from Aldrich and used without further purification. Plant Material. Celastrus vulcanicola J. (Donnell Smith) (Celastraceae) was collected in November 2009 at the Montecristo National Park (2051 m above sea level) in the municipality of Metapán, Province of Santa Ana, El Salvador. The plant material was identified by Jorge Alberto Monterrosa Salomón, and a voucher (J. Monterrosa and R. Carballo 412) specimen is deposited in the Herbarium of Missouri Botanical Garden, St. Louis, MO, USA. Extraction and Isolation. On the basis of previous reports,24 a modified methodology was used for the extraction of the lead compound 1 to optimize the overall yield. Thus, the dried leaves of C. vulcanicola afforded 850.6 mg of compound 1 (0.08% dry weight), the main secondary metabolite in the plant. A detailed description of the extraction process is included in the Supporting Information (Figure S81). Materials for Biological Studies. Vinblastine (VLB), verapamil (VRP), 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and sodium orthovanadate were purchased from SigmaAldrich (Madrid, Spain). Daunomycin (DNM) was purchased from Pfizer (Madrid, Spain). Cell Lines. The parental drug-sensitive NIH-3T3 cell line and one transfected with human MDR1-G185 (MDR1 cells) were cultured in DMEM (Invitrogen, Barcelona, Spain) with 10% heat-inactivated fetal bovine serum (iFBS), containing L-glutamine (2 mM), penicillin G (250 U/mL), and streptomycin (250 μg/mL), as previously described.38 Modulation of DNM Efflux. The DAS studied in the present work were screened for their ability to block the P-gp-dependent DNM efflux by flow cytometry analysis, as previously described.38 Briefly, 24 h before the experiment, MDR1 cells in the logarithmic phase of growth were seeded in 24-well plates at a density of 105 cells per well. Afterward, cells were incubated for 30 min at 37 °C in DMEM + 10% iFBS in the presence or absence of each DAS (10 μM) with DNM (2 μM). Finally, cells were washed twice with ice-cold PBS, trypsinized, and resuspended in 0.2 mL of ice-cold PBS for immediate analysis. Fluorescence measurements of individual cells were made with a Becton Dickinson FacScan (BD European HQ, ErembodegemAalst, Belgium). To calculate Ki values for inhibition of DNM efflux, cells were incubated with DNM as described above in the presence or

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01429. NMR spectra, experimental data and procedures for preparation of analogues 3−46, and 49−82, and inhibition of P-gp-mediated DNM transport and reversal index factor data of selected compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*For S.C.: phone, +34-958-181666; fax, +34-958-181632; Email, [email protected]. *For I.L.B.: phone, +34-922-318594; fax, +34-922-318571; Email, [email protected]. Author Contributions §

O.C. and M.P.S.-C. contributed equally to the present work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the FP7-REGPOT-2012-CT2012316137-IMBRAIN and Fundación CajaCanarias SALUCAN03 projects, the Plan Andaluz de Investigación, Proyecto de Excelencia P08-CTS-03625, and by FEDER funds from the EU (S.C., F.G.), and the Plan Andaluz de Investigación cod. BIO130 (F.G.). We also thank Dr. Ira Pastan (National Cancer Institute, NIH, Bethesda, MD) for providing the NIH-3T3 and NIH-3T3MDR-G185 cell lines.

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ABBREVIATIONS USED MDR, multidrug resistance; ABC, ATP-binding cassette; P-gp, P-glycoprotein; DAS, dihydro-β-agarofuran sesquiterpenes; MDR1, multidrug resistance gene 1; DNM, daunomycin; VLB, vinblastine; VRP, verapamil; RI, reversal index; SAR, structure−activity relationship; DCC, N,N′-dicyclohexylcarbodiimide; DMAP, 4-(N,N-dimethylamino)pyridine; py, pyridine; DMF, dimethylformamide; rt, room temperature; PCC, pyridinium chlorochromate; PBS, phosphate buffered saline; I



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MTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide

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Optimization by Molecular Fine Tuning of Dihydro-β-agarofuran

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