Salinomycin Hydroxamic Acids: Synthesis, Structure, and Biological

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Letter

Salinomycin hydroxamic acids: synthesis, structure, and biological activity of polyether ionophore hybrids. Björn Borgström, Xiaoli Huang, Eduard N. Chygorin, Stina M. Oredsson, and Daniel Strand ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00079 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 26, 2016

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Salinomycin hydroxamic acids: synthesis, structure, and biological activity of polyether ionophore hybrids. Björn Borgström, a Xiaoli Huang, b Eduard Chygorin,a Stina Oredsson, b and Daniel Strand. a * a

Centre for Analysis and Synthesis, Department of Chemistry, Lund University, Box 124, 221 00 Lund, Sweden. bDepartment of Biology, Lund University, Sweden. ABSTRACT: The polyether ionophore salinomycin has recently gained attention due to its exceptional ability to selectively reduce the proportion of cancer stem cells within a number of cancer cell lines. Efficient single step strategies for the preparation of hydroxamic acid hybrids of this compound varying in N– and O–alkylation are presented. The parent hydroxamic acid, salinomycin-NHOH, forms both inclusion complexes and well-defined electroneutral complexes with potassium and sodium cations via 1,3-coordination by the hydroxamic acid moiety to the metal ion. A crystal structure of an cationic sodium complex with a non-coordinating anion corroborates this finding, and moreover reveals a novel type of hydrogen bond network that stabilizes the head-to-tail conformation that encapsulates the cation analogously to the native structure. The hydroxamic acid derivatives display down to single digit µM activity against cancer cells but unlike salinomycin selective reduction of ALDH+ cells, a phenotype associated with cancer stem cells, was not observed. Mechanistic implications are discussed.

Introduction. Amalgamation of structural elements from two or more bioactive compounds into a hybrid structure is an attractive strategy in the design of functional analogs.1,2,3 In principle, such hybrids can expedite syntheses through simplified structures, provide mechanistic insight, and advantageously modulate properties like bioavailability, stability, and selectivity.4,5,6,7,8,9,10,11,12,13,14,15,16,17,18 A structure of interest in this context is the polyether ionophore salinomycin (SA, Figure 1).19 In addition to its industrial use as an anti-coccicide and growth promoter, this compound, and its more active semi-synthetic analogs,20 has been shown to efficiently and selectively inhibit properties associated with breast cancer stem cells (CSC).21,22,23 More recently, SA has also been shown to reduce scarring during wound healing.24 Several studies have been directed at conversion of the carboxylate moiety of this compound and the related monensin into the corresponding esters and amides to impede the ability of electroneutral transport of metal ions across bilayers.25,26,27,28,29,30,31 Particularly secondary amides of monensin display an increased selectivity for relaxing sodium gradients in this context.32 In light of this, we envisioned hydroxamic acid hybrid structures as attractive targets that would combine a novel 1,3-coordination motif capable of binding alkali metal ions like K+ and Na+ with a hydrophobic encapsulation by the SA polyether scaffold. In particular such hybrids would be well suited as probes for investigating a connection between ionophore properties and selective effects against CSCs. Despite the considerable interest in hydroxamic acids in natural product hybrids,33,34,35,36,37,38 as siderophores,39 inhibitors of stat3,40 histone deacetylase (HDAC),41,42 and Ras signaling,43 as well as isosters to carboxylic acids,44 no such analogs has been described for polyether ionophores. A single study by Miller on the pore forming depsipeptide daptomycin highlights two hydroxamic acid derivatives of a natural product with antiporter activity.45 Here, we present efficient pathways relying on direct carboxylate activation to hydroxamic acid analogs of SA systemati-

cally varying in alkylation at the hydroxamate nitrogen and oxygen. Structural and functional investigations of the resulting library reveal down to single digit µM activity against JIMT-1 and MCF-7 breast cancer cells. In contrast to the native structure however, little or no selectivity was found against aldehyde dehydrogenase positive cells (ALDH+), a phenotype associated with CSC properties such as high tumourigenicity and increased migration.46,47 OH O C

B O

O

D O

N H

O N

A

H O

O

Salinomycin (1)

OH N

HN

O

OH

Trichostatin (2)

OH

O HO

O

E

O

H N

O

(CH2)5OH N OH

O

C1 OH HO OH

O

OH O H

Monensin A (3) H O H

O

H

N O

O

H O

OH

O NOH

O HO

OH

O

O

O O

OH

HN OH

HO

OH

OH Salmycin A (4)

Figure 1. Polyether ionophores and hydroxamic acid natural products.

Results and Discussion. Efficient synthesis of unsubstituted hydroxamic acids is challenging, in part due to the hydrolytic sensitivity of many such products.48,49 In light of this and of the low accessibility of the carboxylate moiety of SA,20 it is noteworthy that single step access to each of the targeted hybrid derivatives 5a–d could be attained (Scheme 1). Especially, as a classical method like aminolysis with NH2OH when applied to salinomycin methyl ester failed to give even trace amounts of product. Minor variations in the reaction conditions such as the choice of uronium-type coupling reagent minimized both side product formation and decomposition of the sensitive structures and enabled isolation of each analog by flash chromatography.

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X PF6– HATU: X = 1-hydroxbenzotriazole TFFH: X = F N N TCFH: X = Cl

A:

OH

HATU, DIPEA, NH2OH, DMF, 70%

C20

O

or B: 1. TCFH, DIPEA, NH2OTBS 2. TBAF, THF 40%

O

C28

OH

O

C11

HO

SA–N(H)OH 5a

H O

C1

O N H

OH [scXRD 5a . NaPF6]a,b

O

O

O

O

O

OH O

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OH

OH

O

O

HO

1

H O

O OH

OH

O HO

CH2Cl2, 65%

O

O

O

TFFH, DIPEA, NH2OMe.HCl,

SA–N(H)OMe 5b

H O

O N H

TFFH, DIPEA, MeNHOMe.HCl, CH2Cl2, 76%

O

C1

[scXRD 5b]a

OH O

OH O

HO

CH2Cl2, 36% H O

OH

O

TFFH, DIPEA, MeNHOH.HCl,

OH

O HO

O

O

O

SA–N(Me)OMe 5c

H O

O

O

HO

H N

N

O

OH H O

SA–N(Me)OH 5d O N

6

O

O

O O

N,O-acylotropy O

O

O

O

O

OH

OH

C1 [scXRD 5d]a

TCFH, DIPEA, MeNHOTBS, CH2Cl2 64%

Scheme 1. Systematic synthesis of hydroxamic acid hybrid analogs of 5a–d. a) Thermal ellipsoids are drawn at 30% probability. Selected hydrogen atoms involved in hydrogen bonding are shown. White = hydrogen atom; Red = oxygen atom; Blue = nitrogen atom; Purple = sodium atom. b) The PF6– counter ion and a solvate EtOAc are removed for clarity.

The parent compound in the series, hydroxamic acid 5a was synthesized with complete selectivity for N-acylation by reacting SA with free base hydroxylamine in DMF using HATU as the coupling reagent. A two-step protocol via formation of an O-tertbutyldimethylsilyl (TBS) protected intermediate followed by removal of the silyl group with TBAF was also evaluated but gave lower yields over the two steps. Despite extensive experimentation, we were unable to crystallize 5a or its Na+ or K+ salts. Drawing on a report of a neutral NaClO4 complex of monensin however,50,51 we were gratifyingly able to obtain single crystals of a neutral form of 5a binding a sodium atom with a non-coordinating PF6– counter ion. On a general note, this result suggests that alkali metals with non-coordinating counter ions may be of a broader utility for growing crystals of related compounds that are otherwise difficult to crystallize. The scXRD structure of 5a.NaPF6 corroborated the structural assignment and moreover the premise that such derivatives are capable of neutral metal cation coordination via 1,3binding of the cation by the hydroxamic acid moiety. Similarly to the crystal structure of SA.Na,52 5a.NaPF6 shows coordination to the metal ion from the oxygen atoms of the D- and E-rings as well as from the C11 carbonyl group. The acyl group in 5a.NaPF6 however displays a strikingly different behavior from that of the carboxylate of SA.Na. The head-to-tail conformation in SA.Na is stabilized by hydrogen bonds from the C9 and C28 hydroxyl groups to the carboxylate, which are absent in 5a.NaPF6. Instead, a novel type of hydrogen bond network stabilizes a similar conformation wherein

the C20 hydroxyl group serves as hydrogen bond donor53 to the hydroxamate OH (O…O = 2.9 Å) that in turn donates a hydrogen bond to the C28 oxygen atom (O…O = 2.6 Å). The Weinreb amide 5c and the methyl hydroxamate ester 5b were both straightforwardly synthesized using the more reactive TFFH coupling reagent. Access to the final N-methyl analog 5d proved more challenging; under the conditions used for 5b and 5c, coupling with N-methyl hydroxyl amine gave a 3:1 mixture of azanyl ester 6 and N-methyl hydroxamic acid 5d from which 6 was isolated in a 36% yield. The connectivity of this compound was established by a 15N HSQC correlation from the acidic NH to nitrogen. Over time, a methanolic solution of azanyl ester 6 rearranged to the desired N-methyl-hydroxamic acid 5d, a type of reactivity that was noted by Jencks and later Nikishin.54,55 A more practical synthesis of 5d was accomplished using O-TBS protected N-methyl hydroxylamine with TCFH as the coupling reagent. Interestingly, the silyl group spontaneously hydrolyzed during the reaction/isolation process, enabling direct isolation also of 5d. Single crystals of both 5b and 5d were obtained by recrystallization from EtOAc/n-heptane and the scXRD structures of both compounds parallel that of the original p-iodophenacyl ester of salinomycin.56 An unusual s-trans conformation of the hydroxamate group of 5d is stabilized by a hydrogen bond between the terminal hydroxyl group and the C9 oxygen.

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The biological activity of SA is intrinsically connected to its ability to bind, exchange, and release ions. The ability of the hybrid structures 5a–d to coordinate and exchange alkali metal ions was therefore investigated (Scheme 2). As expected, washing an EtOAc solution of 5a with aqueous K2CO3 or Na2CO3 provided homogenous and well-defined metal cation complexes shown by a diagnostic disappearance of the acidic proton (δ = 10.3) in the 1H NMR spectrum in benzene-d6. OH O

O

O

O OH

O

O HO

K+ N H

O– 7

H O

O N H

5a

.) N (s aC at l .a q

OH O

HO

H O

M+ ClO N H

O

OH 5a ⋅ MCl

10.0 ± 0.4

SA–N(H)OMe 5b

17.3 ± 0.7

16.3 ± 0.9

SA–N(Me)OMe 5c

10.1 ± 0.4

9.3 ± 1.7

(>20)

(>20)

0.52 ± 0.09

0.59 ± 0.08

b

HCl (0.1 M aq.)

OH

O HO

Figure 2. ALDEFLUOR assay of derivatives 5a-d.a

O

O

O Na2CO3 (0.1 M aq.)

OH

O

OH

O

O

O

6.7 ± 0.7

a) MTT-based dose-response assay. MTT reduction is assumed to be proportional to the cell number. IC50 values are the mean (±SE) for 50% reduction compared to control. For all entries n ≥ 3. b) The compound was not fully soluble in DMSO at the concentrations evaluated.

Na2CO3 (0.1 M aq.)

) O 3 q. C a a2 M N .1 (0

HCl (0.1 M aq.)

SA–N(H)OH 5a

Salinomycin (ref. 20)

m

.)

K2CO3 (0.1 M aq.)

MCF-7cells IC50 [µM]

SA–N(Me)OH 5d

OH

4

H O

K2CO3 (0.1 M aq.)

OH

l-d

HO

JIMT-1 cells IC50 [µM]

no

O

Table 1. Antiproliferative activity of derivatives 5a-d. a

O

O

O

et ha

O

JIMT-1 cells, as opposed to the cell surface markers CD44/CD24.23 In contrast to SA and its more active 20-O-acylated analogs, which give a ~60% reduction of ALDH+ cells at the respective IC50 concentration, the hydroxamic acid derivatives 5a–d gave, when evaluated at two concentrations (5 and 10 µM), no or only minor selectivity against this phenotype.

OH

O 3 aq C M K 2 .1 (0

H O

O

Na+ N H

O– 8

Scheme 2. Ion exchange properties of hydroxamic acid 5a. Interconversion of 7 and 8 was readily accomplished using the same protocol. In contrast to salinomycin however, washing an EtOAc solution of 7 or 8 with 0.1 M HCl (aq.) did not result in dissociation of the metal ion. Instead, structures tentatively assigned as inclusion complexes of MCl were formed. Broadened signals in the 1H NMR spectra indicate chemical exchange, but despite extensive experimentation, we could not obtain 5a from 5a.MCl by acid wash. Deprotonation with aqueous carbonates however cleanly returned 7 and 8, and 5a.NaCl was readily and reversibly converted to 5a.KCl. In addition, the inclusion complexes give identical 1H NMR spectra to 5a in methanol-d4 suggesting that dissociation does occur when the complex is dissolved in a protic solvent (See SI for details). The latter is interesting as it suggests that alkali metal ion exchange is viable in biological systems despite a considerable affinity of 5a towards such species. None of the structures 5b–d was deprotonated or appeared to form inclusion complexes under the same conditions. It is also worth noting that both the sensitive salinomycin framework and the hydroxamic acid motifs endured exposure to these acidic and basic conditions without observable decomposition. The cytotoxicity of compounds 5a-d was evaluated against two breast cancer cell lines, JIMT-1 and MCF-7, using an MTT based assay (Table 1). The hydroxamic acid derivatives are less active than SA itself, but showed IC50’s down to the single digit µM range. The capacity of 5a–d to selectively reduce traits associated with cancer stem cells was evaluated using an ALDEFLUOR assay (Figure 2). This assay has previously been used to assess phenotype selectivity based on the expression of aldehyde dehydrogenase (ALDH). This assay gives linear dose dependent responses in

a) ALDEFLUOR assay for CSC selectivity in JIMT-1 cells. Reduction in the proportion of ALDH+ cells compare to control, reported as the mean (±SE). The proportion of ALDH+ in untreated cells was 30–60%. For all entries n ≥ 3.

Figure 3. H+/K+ exchange across a lipid membrane mediated by derivative 5a-d and salinomycin.a 5c (1 pM) 5b/c (1 pM)

7.5

5a (1 pM)

pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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SA 1 (1 pM) 5a (100 pM) 7 0

100

200

300

Time (s)

a) Ion exchange was monitored by the acidification inside large unilamellar vesicles (lipid ratio POPC/POPG 3:1) as determined by the pH reporter pyranin.

In line with this result, 5a requires over 100-fold higher concentrations to show similar activity in relaxing alkali metal ion gradients across membranes of large unilamellar vesicles compared to SA (Figure 3, see Supporting Information for details). Our interpretation of the lack of selectivity against ALDH+ cells, even at concentrations where cell proliferation is considerably impeded, is that the primary mechanism of toxicity exhibited by the hydroxamic acid hybrids is altered from that of SA.57 These findings are in line

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with our previous studies where only salinomycin analogs with a free carboxylic acid give phenotype selectivity.22 Conclusions. In conclusion, efficient single step methods for preparation of a library of hydroxamic acid hybrids of the polyether ionophore SA are presented. The products exemplify a novel type of hybrid structure between hydroxamic acids and polyether ionophores, and include the first example of a C1-modified analog of SA with a demonstrated ability to form both well-defined neutral complexes with alkali metal ions and inclusion complexes with NaCl and KCl. An scXRD structure of hydroxamic acid 5a exhibits a novel type of hydrogen bond network that stabilizes a head-to-tail conformation, which encapsulates the bound metal ion. Metal ion exchange- and transport experiments further show that such complexes can participate in metal ion translocation across biological membranes, although less efficiently than the native structure. The hydroxamic acid derivatives exhibit low µM activities against breast cancer cells, but unlike the native structure, little to no selectivity for reduction of the proportion of ALDH+ cells, a phenotype associated with CSC properties, was found. Mechanistically, the results point to a different primary origin of the observed cytotoxicity effects by such derivatives compared to that of SA. Additionally the results underscore that the phenotype selectivity of salinomycin appears to originate from a capacity for efficient ion transport. Further studies on these and related structures against CSCs and as

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mechanistic probes are currently under way and will be reported in due course.

ASSOCIATED CONTENT Supporting Information Experimental procedures and characterization for compounds 5a, 5b, 5c, 5d, 6, 5a.NaPF6, 7, and 8. Structure elucidation of all compounds. Copies of 1H and 13C NMR spectra for all new compounds. scXRD data for 5a, 5b, and 5d. (Accession Codes. CCDC: 1446108, 1446121, 1446122) Procedures for ion exchange and transport experiments. Experimental details of the MTT and ALDEFLUOR assays. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]

ACKNOWLEDGMENT We thank the Swedish research council (VR), the Crafoord foundation, The Royal Swedish Academy of Sciences (KVA), and the Swedish Cancer Society (CF) for financial support. We thank Dr. H. v. Wachenfeldt for 2D NMR experiments.

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Efficient new constructs against triple negative breast cancer cells: synthesis and preliminary biological study of ferrocifen–SAHA hybrids and related species. Dalton Trans. 2013, 42, 15489–15501. 37 Jiang, B.; Huang, X.; Yao, H.; Jiang, J.; Wu, X.; Jiang, S.; Wang, Q.; Lue, T.; Xu, J. Discovery of potential anti-inflammatory drugs: diaryl1,2,4-triazoles bearing N-hydroxyurea moiety as dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase Org. Biomol. Chem. 2014, 12, 2114– 2127. 38 Bogatyrenko, T. N.; Konovalova, N. P.; Sipyagin, A. M.; Bogatyrenko, V. R.; Kuropteva, Z. V.; Baider, L. M.; Sashenkova, T. E.; Fedorov, B. S. Polyfunctional action of biologically active compounds in antitumor chemotherapy of cyclophosphamide. Russ. Chem. Bull. 2014, 63, 1187–1191. 39 For lead reference see: Miller, M. J. Syntheses and therapeutic potential of hydroxamic acid based siderophores and analogs. Chem. Rev. 1989, 89, 1563–1579. 40 Yue, P.; Lopez-Tapia, F.; Paladino, D.; Li, Y.; Chen, C-.H.; Hilliard, T.; Chen, Y.; Tius, M. A.; Turkson J. Hydroxamic Acid and Benzoic Acid–Based STAT3 Inhibitors Suppress Human Glioma and Breast Cancer Phenotypes In Vitro and In Vivo. Cancer Res. published online June 18, 2015; DOI: 10.1158/0008-5472.CAN-14-3558. 41 Marks, P. A.; Rifkind, R. A.; Richon, V. M.; Breslow, R.; Miller, T.; Kelly, W. K. Histone deacetylases and cancer: causes and therapies. Nature Rev. Cancer 2001, 1, 194–202.

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42 For a study of combination effects from hydroxamic acid HDAC inhibitor and SA, see: Booth, L.; Roberts, J. L.; Conley, A.; Cruickshanks, N.; Ridder, T.; Grant, S.; Poklepovic, A.; Dent, P. HDAC inhibitors enhance the lethality of low dose salinomycin in parental and stem-like GBM cells. Cancer Biol. Ther. 2014, 15, 305–316. 43 Patel, D. V.; Young, M. G.; Robinson, S. P.; Hunihan, L.; Dean B. J.; Gordon E. M. Hydroxamic acid-based bisubstrate analog inhibitors of ras farnesyl protein transferase. J. Med. Chem. 1996, 39, 4197–4210. 44 Ballatore, C.; Huryn, D. M.; Smith, A. B. III Carboxylic acid (bio)isosteres in drug design ChemMedChem 2013, 8, 385–395. 45 Yoganathan, S.; Yin, N.; He, Y.; Mesleh, M. F.; Gu, Y. G.; Miller, S. J. An efficient chemical synthesis of carboxylate-isostere analogs of daptomycin. Org. Biomol. Chem. 2013, 11, 4680–4685. 46 Xu, X.; Chai, S.; Wang, P.; Zhang, C.; Yang, Y.; Yang, Y.; Wang, K. Aldehyde dehydrogenases and cancer stem cells. Cancer. Lett. 2015, 369, 50-57. 47 Ginestier, C.; Hur, M. H.; Charafe-Jauffret, E.; Monville, F.; Dutcher, J.; Brown, M.; Jacquemier, J.; Viens, P.; Kleer, C. G.; Liu, S.; Schott, A.; Hayes, D.; Birnbaum, D.; Wicha, M. S.; Dontu, G. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 2007, 1, 555–567. 48 For hydroxamic acid syntheses, see ref 48-49: Porcheddu, A.; Giacomelli, G. Synthesis of oximes and hydroxamic Acids. In The chemistry of hydroxylamines, oximes and hydroxamic acids. Rappapport, Z.; Liebman Eds. Wiley-VCH Verlag GmbH. L. F. Weinheim, Germany; 2009, pp. 163–231. 49 Yale, H. L. The hydroxamic acids. Chem. Rev. 1943, 33, 209–256 and references therein. 50 Ward, D. L.; Wei, K-.T.; Hoogerheide J. G.; Popov A. I. The crystal and molecular structure of the sodium bromide complex of monensin, C36H62O11.Na+Br−. Acta Cryst. B, 1978, 34, 110–115. 51 Huczyński A.; Janczak J.; Łowicki D.; Brzezinski B. Monensin A acid complexes as a model of electrogenic transport of sodium cation. Biochim. Biophys. Acta, Biomembr. 2012, 1818, 2108–2119. 52 Paulus, E. F.; Kurz, M.; Matter, H.; Vértesy L. Solid-state and solution structure of the salinomycin−sodium complex:  Stabilization of different conformers for an ionophore in different environments. J. Am. Chem. Soc. 1998, 120, 8209-8221. 53 This hydroxyl group is not involved in metal ion coordination or in hydrogen bonding to the acid motif of SA. 54 Jenks, W. P. The reaction of hydroxylamine with activated acyl groups. I. Formation of O-acylhydroxylamine. J. Am. Chem. Soc. 1958, 80, 4585–4588. 55 Nikishin, G. I.; Troyansky, E. I.; Svitanko, I. V.; Chizhov, O. S. N,O-acylotropy in N-methylpentanehydroxamic acid. Tetrahedron Lett. 1984, 25, 97–98. 56 Kinashi, H.; Ōtake, N.; Yonehara, H. Studies on the ionophorous antibiotics I. The crystal and molecular structure of salinomycin piodophenacyl ester. Acta Cryst. 1975, B31, 2411-2415. 57 Similar levels of toxicity is also found for C1-esters and amide derivatives of SA, see references 20, 22, and 25-29 respectively.

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Salinomycin hydroxamic acids: synthesis, structure, and biological activity of polyether ionophore hybrids. Björn Borgström, a Xiaoli Huang, b Eduard Chygorin,a Stina Oredsson, b and Daniel Strand. a *

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