Elsevier

Bioorganic & Medicinal Chemistry

Volume 16, Issue 2, 15 January 2008, Pages 1015-1031
Bioorganic & Medicinal Chemistry

Novel analogs of d-e-MAPP and B13. Part 1: Synthesis and evaluation as potential anticancer agents

https://doi.org/10.1016/j.bmc.2007.08.033Get rights and content

Abstract

A series of novel isosteric analogs of the ceramidase inhibitors, (1S,2R)-N-myristoylamino-phenylpropanol-1 (d-e-MAPP) and (1R,2R)-N-myristoylamino-4′-nitro-phenylpropandiol-1,3 (B13), with modified targeting and physicochemical properties were designed, synthesized, and evaluated as potential anticancer agents. When MCF7 cells were treated with the analogs, results indicated that the new analogs were of equal or greater potency compared to the parent compounds. Their activity was predominantly defined by the nature of the modification of the N-acyl hydrophobic interfaces: N-acyl analogs (class A), urea analogs (class B), N-alkyl analogs (class C, lysosomotropic agents), and ω-cationic-N-acyl analogs (class D, mitochondriotropic agents). The most potent compounds belonged to either class D, the aromatic ceramidoids, or to class C, the aromatic N-alkylaminoalcohols. Representative analogs selected from this study were also evaluated by the National Cancer Institute In Vitro Anticancer Drug Discovery Screen. Again, results showed a similar class-dependent activity. In general, the active analogs were non-selectively broad spectrum and had promising activity against all cancer cell lines. However, some active analogs of the d-e-MAPP family were selective against different types of cancer. Compounds LCL85, LCL120, LCL385, LCL284, and LCL204 were identified to be promising lead compounds for therapeutic development.

Introduction

Cancer has proven to be a difficult disease to treat, and few effective drugs are available. Identification of novel, efficient, selective, and less toxic anticancer agents remains an important and challenging goal in medicinal chemistry.1, 2, 3

Sphingolipid (SPL) metabolism provides a rich network of bioactive molecules that play key roles in the regulation of diverse cell functions. Ceramide (Cer) has emerged as a key modulator of cancer cell growth and apoptosis.4 Sphingosine 1-phosphate (S1P), generated from Cer by the action of ceramidases (CDases) to yield sphingosine (Sph) which is phosphorylated by sphingosine kinase (SK), promotes growth and opposes Cer-mediated apoptosis.5, 6 Because of the roles of Cer and S1P in regulating cell growth and cell death, their metabolic and signaling pathways have emerged as potential targets for anticancer therapy.7 Many approaches have been explored to increase endogenous Cer, and a few appear promising.7, 8 These include the application of cell permeable short-chain Cers,9, 10, 11 liposomal formulations,12, 13 site-specific cationization,14, 15, 16, 17 and induction of endogenous Cer by modulation of SPL metabolizing enzymes.18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34

In the course of our investigation of SPLs’ chemistry and the search for new molecules that mimic the action of SPLs and/or regulate their metabolism, we have focused on analogs that affect Cer metabolism, especially the Cer-Sph-S1P balance, and display particular desired properties, and we have also attempted to target them to specific sub-cellular compartments.14, 15, 16, 17, 27, 28, 31

Previously, we synthesized a set of lipophilic phenyl-N-acyl-amino-alcohols (aromatic analogs of Cer, Scheme 1) and evaluated their structure-dependent inhibitory effects in HL-60 cells.20, 21 We found that analog activity depended on stereochemistry and the chain length of the N-acyl moiety. Analogs with the natural Cer configuration were not active compounds, whereas their stereoisomers inhibited cell growth. The active isomers were N-dodecanoyl (C12), N-myristoyl (C14), and N-palmitoyl (C16) homologs, with C14-homologs being the most potent. Shorter (C2–C10) and longer (C18–C24) homologs were considerably less active. Pilot modification of some N-myristoyl analogs (amides) to their N-myristyl-counterpart (N-alkyl-amines) increased their inhibitory activity in HL-60 cells (A. Bielawska, data not published).

Further investigations on the active analogs confirmed their inhibitory effects on ceramidases (CDases). HL-60 cell experiments and in vitro studies showed that the most potent analogs from this group, d-e-MAPP, stereospecifically inhibited alkaline CDase, whereas its enantiomer, l-e-MAPP, served as a substrate for this enzyme.20 Biological activity of d-e-MAPP was later confirmed by several investigators.35, 36, 37, 38, 39, 40 Another active analog: B13 (Scheme 1), which differs from d-e-MAPP in stereochemistry and functional groups, inhibited acid CDase.32, 34 Treatment with B13 caused the release of cytochrome C and induced apoptosis.34 Biological activity of B13 was also demonstrated in malignant melanoma, colon, and prostate cancer cells, and in animal experiments of in vivo cancer growth.32, 33, 34

d-e-MAPP, B13, and Cers are N-acylamino alcohols with two distinct elements of rigidity: a phenyl ring or long chain alkenyl unit and the amido group joined to the chiral backbone of the propane chain (Scheme 1). These structural features determine their conformation(s) and ability to interact effectively with putative targets, via hydrogen bonding and van der Waals hydrophobic interactions. Therefore, the aromatic analogs of Cer constitute attractive models for the development of new bioactive lipophilic molecules. The effects of additional aromatic analogs on SPL metabolism and SPL intracellular trafficking have been reported, demonstrating that the phenyl-amino-alcohol foundation is recognized by many enzymes of SPL metabolism and serves as a useful structural benchmark for development of bioactive compounds.18, 19, 22, 23, 24, 25, 26, 29, 41

In a previous study, we showed that isosteric replacement of the amide group of Cer by urea or N-alkylamine units generated inhibitors of neutral CDase, thus illustrating the usefulness of this approach.42 Moreover, in another recent study, we developed the concept of the fixed positive charge-dependent cellular targeting Cer, and demonstrated that fixed cationic Cer analogs target preferentially to the mitochondria.14, 16, 17

Extending these findings to the aromatic analogs of Cer, we suspected that introduction of specific structural features and other modifications of −CO, X, Y, R, and R1-functionalities as well as altering the stereochemistry of d-e-MAPP and B13 structures (Scheme 1) will improve and modify their physicochemical and targeting properties to specific compartments.

These new compounds are shown in Scheme 2. Based on targeting behavior of alkylamines, we expected that some analogs will locate to lysosomes (e.g., N-alkylamino analogs, class C).43, 44, 45, 46 In contrast, fixed cations are expected to act as a mitochondriotropic agents (aromatic ceramidoids, class D).17 Finally, neutral analogs (parent amides, N-methyl amides, class A, and urea analogs, class B) may show no compartmental preferences as was shown for exogenous Cers.47, 48

Here we describe the synthesis, full NMR characterization, basic physicochemical properties, and growth suppression of breast carcinoma MCF7 cells of new analogs of d-e-MAPP and B13. Additionally, we present results from the National Cancer Institute for a full screening against a 60-human-tumor-cell assay (the NCI’s 60-cell line assay) performed for the selected analogs from classes A–D.

Section snippets

Chemistry

The compounds designed and synthesized in this study represent the second generation of analogs that are based on structural and stereochemical modifications of d-e-MAPP and B13 (Scheme 1). The selected model compounds generally contain C14-hydrocarbon chains in their N-acyl parts. Structural analogs contain a modified proton donor–acceptor ability of the functional groups at C1-, C2-, and C3-positions and substituents of the phenyl ring. The stereochemical isomers are: (1R,2S), (1R,2R), (1S,2R

Summary and conclusions

Based on the chemical structure differences and cell activity presented here, compounds were grouped into 4 classes (A, B, C, and D). Class A: N-acyl analogs (neutral); class B: urea analogs (neutral); class C: N-alkyl analogs (lysosomotropic analogs); class D: ω-cationic analogs (mitochondriotropic analogs).

All new analogs showed an increased inhibitory effect on MCF7 cell growth, compared to their parent compounds. Class A and class B were active with IC50 > 10 μM, whereas analogs from classes C

Chemistry

All solvents, general reagents, and starting amino alcohols 1–13 (Scheme 2) were purchased from Aldrich-Sigma & Fluka. d-e-MAPP, l-e-MAPP, (1R,2R)-B13, and (1S,2S)-B13 were prepared from the corresponding amino alcohols 5, 6, 10 and 11 as described previously.20, 21 Purity of these compounds was confirmed by 1H NMR and the optical rotation values: d-e-MAPP: [α]D23+14.1 (c 1, MeOH); [α]36523+43.9 (c 1, MeOH); l-e-MAPP: [α]D23-14.9 (c 1, MeOH); [α]36523-45.6 (c 1, MeOH). (1R,2R)-B13: [α]D24-29.3 (

Acknowledgments

Financial support was provided by the NIH NCI—PO1CA097132. Special acknowledgment is for NCRR—CO6RR018823 providing laboratory space for Lipidomics Core in CRI building. We thank Dr. Jennifer Schnellmann for editorial assistance.

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