Synthesis and In Vitro Activity of Novel Melphalan Analogs in Hematological Malignancy Cells.

Despite the continuous developments in pharmacology and the high therapeutic effect of new treatment options for patients with hematological malignancies, these diseases remain a major health issue. Our study aimed to synthesize, analyze in silico, and determine the biological properties of new melphalan derivatives. We obtained three methyl esters of melphalan having in their structures amidine moieties substituted with thiomorpholine (EM-T-MEL), indoline (EM-I-MEL), or 4-(4-morpholinyl) piperidine (EM-MORPIP-MEL). These have not yet been described in the literature. The in vitro anticancer properties of the analogs were determined against THP1, HL60, and RPMI8226 cells. Melphalan derivatives were evaluated for cytotoxicity (resazurin viability assay), genotoxicity (alkaline comet assay), and their ability to induce apoptosis (Hoechst33342/propidium iodide double staining method; phosphatidylserine translocation; and caspase 3/7, 8, and 9 activity measurements). Changes in mitochondrial membrane potential were examined using the specific fluorescence probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol carbocyanine). The EM-T-MEL derivative had the highest biological activity, showing higher cytotoxic and genotoxic properties than the parent drug. Moreover, it showed a high ability to induce apoptosis in the tested cancer cells. This compound also had a beneficial effect in peripheral blood mononuclear cells (PBMC). In conclusion, we verified and confirmed the hypothesis that chemical modifications of the melphalan structure improved its anticancer properties. The conducted study allowed the selection of the compound with the highest biological activity and provided a basis for chemical structure-biological activity analyses.

Biodegradable reduction-responsive polymeric micelles for enhanced delivery of melphalan to retinoblastoma cells.

Melphalan (MEL) is an effective chemotherapeutic agent for treatment of retinoblastoma (Rb) which is the most common childhood malignancy. However, the inherent cardiopulmonary toxicity and hazardous integration limit its therapeutic effect on RB. N-Acetylheparosan (AH), a natural heparin-like polysaccharide in mammals with long circulation effect and good biocompatibility, was linked by d-α-tocopherol acid succinate (VES) via and cystamine (CYS) to synthesize reduction-responsive N-acetylheparosan-CYS-Vitamin E succinate (AHV) copolymers. In addition, CYS was replaced by adipic acid dihydrazide (ADH) to obtain a control of non-reduction-responsive polymers N-acetylheparosan-ADH-Vitamin E succinate (ADV). MEL-loaded AHV micelles (MEL/AHV) as well as ADV micelles (MEL/ADV) were prepared with small particle size and high drug loading content. In vitro drug release showed that MEL/AHV micelles presented obvious reduction-triggered release behavior compared with MEL/ADV. In vitro antitumor effects were investigated using WERI-Rb-1 retinoblastoma cells. Cytotoxicity experiments showed that the IC50 of MEL/AHV was significantly lower than that of free MEL and MEL/ADV, suggesting that MEL/AHV enhanced the cytotoxicity against retinoblastoma cells. Furthermore, MEL/AHV micelles were more easily uptaken by multiple pathways compared with MEL/ADV and free MEL. Therefore, MEL/AHV might be a potential delivery system for enhanced delivery of melphalan to Rb cells.

N-Alkyl-1,5-dideoxy-1,5-imino-l-fucitols as fucosidase inhibitors: Synthesis, molecular modelling and activity against cancer cell lines.

1,5-Dideoxy-1,5-imino-l-fucitol (1-deoxyfuconojirimycin, DFJ) is an iminosugar that inhibits fucosidases. Herein, N-alkyl DFJs have been synthesised and tested against the α-fucosidases of T. maritima (bacterial origin) and B. taurus (bovine origin). The N-alkyl derivatives were inactive against the bacterial fucosidase, while inhibiting the bovine enzyme. Docking of inhibitors to homology models, generated for the bovine and human fucosidases, was carried out. N-Decyl-DFJ was toxic to cancer cell lines and was more potent than the other N-alkyl DFJs studied.

Stereoselective synthesis and antiproliferative activity of the isomeric sphinganine analogues.

A flexible synthetic approach to biologically active sphingoid base-like compounds with a 3-amino-1,2-diol framework was achieved through a [3,3]-sigmatropic rearrangement and late stage olefin cross-metathesis as the key transformations. The stereochemistry of the newly created stereogenic centre was assigned via a single crystal X-ray analysis of the (4S,5R)-5-(hydroxymethyl)-4-vinyloxazolidine-2-thione. In order to rationalise the observed stereoselectivity of the aza-Claisen rearrangement, DFT calculations were carried out. The targeted isomeric sphingoid bases were screened in vitro for anticancer activity on a panel of seven human malignant cell lines. Cell viability experiments revealed that C17-homologues are more active than their C12 congeners.

Discovery of IDO1 and DNA dual targeting antitumor agents.

The development of small molecules for cancer immunotherapy is highly challenging and indoleamine 2,3-dioxygenase 1 (IDO1) represents a promising target. Inspired by the synergistic effects between IDO1 inhibitors and traditional antitumor chemotherapeutics, the first orally active dual IDO1 and DNA targeting agents were designed by the pharmacophore fusion strategy. The bifunctional hybrids exhibited enhanced IDO1 enzyme inhibitory activity and in vitro cytotoxicity as compared to IDO1 inhibitor 1-methyl-tryptophan and DNA alkylating agent melphalan. In a murine LLC tumor model, the dual targeting agents demonstrated excellent antitumor efficacy, highlighting the advantages of this novel design strategy to improve the efficacy of small molecule cancer immunotherapy.

Synthesis of dual-action parthenolide prodrugs as potent anticancer agents.

Cancer stem cells are responsible for the failure of a large number of cancer treatments and the re-emergence of cancer in patients. Parthenolide is a potent anticancer sesquiterpene lactone that is also able to kill cancer stem cells. The main problem with this compound is its poor solubility in water. To solve this problem, medicinal chemists have tried to prepare amino-derivatives of parthenolide, however, most amino-derivatives have less potency than that of parthenolide. In this paper, we proposed a new approach to synthesize parthenolide derivatives with better solubility and higher potency. We prepared novel parthenolide derivatives through the aza-Michael addition of nitrogen-containing anticancer drug molecules (cytarabine and melphalan) to the α-methylene-γ-lactone group of parthenolide. Different types of catalysts were used to catalyze the aza-Michael addition. Among all the used catalysts, 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) was found to have the highest catalytic activity. In addition, we examined the effects of parthenolide-anticancer drug hybrids on the growth and proliferation of three cancer cell lines (MCF-7, LNcaP, Hep G2) and CHO. The parthenolide prodrugs showed potent cytotoxic property with IC50 values ranging from 0.2 to 5.2µM, higher than those of parthenolide and anticancer drugs (cytarabine and melphalan).

Oxime-based inhibitors of glucose transporter 1 displaying antiproliferative effects in cancer cells.

An analysis of the main pharmacophoric features present in the still limited number of inhibitors of glucose transporter GLUT1 led to the identification of new oxime-based inhibitors, which proved to be able to efficiently hinder glucose uptake and cell growth in H1299 lung cancer cells. The most important interactions of a representative inhibitor were indicated by a novel computational model of GLUT1, which was purposely developed to explain these results and to provide useful indications for the design and the development of new and more efficient GLUT1 inhibitors.

Inhibition of carbonic anhydrase activity modifies the toxicity of doxorubicin and melphalan in tumour cells in vitro.

Carbonic anhydrase IX (CA IX) is a hypoxia-regulated enzyme, overexpressed in many types of human cancer. CA IX is involved in pH homeostasis, contributing to extracellular acidification and tumourigenesis. Acidification of the extracellular milieu can impact upon cellular uptake of chemotherapeutic drugs by favouring weak acids (e.g. melphalan), but limiting access of weak bases (e.g. doxorubicin). We investigated whether alterations of CA IX activity affected anti-cancer drug uptake and toxicity. CA inhibitor acetazolamide (AZM) enhanced doxorubicin toxicity but reduced melphalan toxicity in cell lines that highly expressed CA IX under anoxic conditions (HT29 and MDA435 CA9/18). The toxicity changes reflected modification of passive drug uptake. AZM did not alter toxicity or uptake in cells with low CA IX activity (HCT116 and MDA435 EV1). AZM lowered intracellular pH in HT29 and MDA435 CA9/18 cells under anoxic conditions. CA IX activity has chemomodulatory properties and is an attractive target for anti-cancer therapy.

Bivalent bendamustine and melphalan derivatives as anticancer agents.

The alkylating agents bendamustine and melphalan are currently used in the treatment of various tumoral diseases. In order to increase their antitumor potency and tumor selectivity both compounds were integrated in structure-activity relationship studies including new drug carrier systems. Here we describe the synthesis and the cytotoxicity of new bivalent bendamustine and melphalan derivatives. Two molecules each esterified with N-(2-hydroxyethyl)maleimide were connected by diamines with various chain lengths (n=6, 7, 8, 12). It was supposed that these conjugates (5a-d, 10a-d, 11a-d) cause cytotoxic effects preferred as bivalent drug. Indeed the cytotoxicity of the new compounds increased compared to bendamustine and melphalan as determined in concentration-dependent in vitro assays using the human MCF-7 and MDA-MB-231 breast cancer cell lines.

Novel melphalan and chlorambucil derivatives of 2,2,6,6-tetramethyl-1-piperidinyloxy radicals: synthesis, characterization, and biological evaluation in vitro.

A series of spin-labeled melphalan and chlorambucil derivatives, coupling the alkylating agents with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) radicals, were synthesized, characterized, and their biological properties in vitro were evaluated. These compounds showed much higher cytotoxic activity against human leukemia cell line K562 in vitro than their parent compounds.

Amidine analogues of melphalan: synthesis, cytotoxic activity, and DNA binding properties.

Design, synthesis, and cytotoxic activity of amidine derivatives of melphalan are described and structure-activity relationships are discussed. Evaluation of the cytotoxicity of these compounds employing a MTT assay and inhibition of [(3)H]thymidine incorporation into DNA in both MDA-MB-231 and MCF-7 human breast cancer cells demonstrated that these compounds were more active than melphalan. Data from the ethidium displacement assay showed that these compounds were able to bind in the minor groove-binding mode in AT sequences of DNA. The cytotoxic properties of the amidine analogues of melphalan towards cultured human breast cancer cells correlate with topoisomerase II inhibitory properties but not with DNA-binding properties.

Novel amidine analogue of melphalan as a specific multifunctional inhibitor of growth and metabolism of human breast cancer cells.

A novel amidine analogue of melphalan (AB4) was compared to its parent drug, melphalan in respect to cytotoxicity, DNA and collagen biosynthesis in MDA-MB-231 and MCF-7 human breast cancer cells. It was found that AB4 was more active inhibitor of DNA and collagen synthesis as well more cytotoxic agent than melphalan. The topoisomerase I/II inhibition assay indicated that AB4 is a potent catalytic inhibitor of topoisomerase II. Data from the ethidium displacement assay showed that AB4 intercalated into the minor-groove at AT sequences of DNA. The greater potency of AB4 to suppress collagen synthesis was found to be accompanied by a stronger inhibition of prolidase activity and expression compared to melphalan. The phenomenon was related to the inhibition of beta(1)-integrin and IGF-I receptor mediated signaling caused by AB4. The expression of beta(1)-integrin receptor, as well as Sos-1 and phosphorylated MAPK, ERK(1) and ERK(2) but not FAK, Shc, and Grb-2 was significantly decreased in cells incubated for 24h with 20 microM AB4 compared to the control, not treated cells, whereas in the same conditions melphalan did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. These results indicate the amidine analogue of melphalan, AB4 represent multifunctional inhibitor of breast cancer cells growth and metabolism.

Prolidase, a potential enzyme target for melanoma: design of proline-containing dipeptide-like prodrugs.

Bioinformatics tools such as Perl, Visual Basic, Cluster, and TreeView were used to analyze public gene expression databases in order to identify potential enzyme targets for prodrug strategies. The analyses indicated that prolidase might be a desirable enzyme target based on its differential expression in melanoma cancer cell lines and its high substrate specificity for dipeptides containing proline at the carboxy terminus. RT-PCR expression of prolidase and hydrolytic activity against N-glycyl-l-proline (GLY-PRO), a standard substrate of prolidase, determined in tumor cell lines, exhibited a high correlation (r(2) = 0.95). These results suggest the possibility of targeting prolidase with prodrugs of anticancer agents for enhanced selectivity. The feasibility of such a scenario was tested by (a) synthesizing prodrugs of melphalan that comprised linkage of the carboxy terminus of the l-phenylalanine moiety of melphalan to the N-terminus of l and d stereoisomers of proline and (b) determining their bioconversion and antiproliferative activities in SK-MEL-5 cells, a melanoma cancer cell line with high expression levels of prolidase. The results of hydrolysis studies of the l- and d-proline prodrugs of melphalan, designated as prophalan-l and prophalan-d, respectively, indicated a approximately 7-fold higher rate of activation of prophalan-l compared to prophalan-d in SK-MEL-5 cell homogenates. Prophalan-l exhibited cytotoxicity (GI(50) = 74.8 microM) comparable to that of melphalan (GI(50) = 57.0 microM) in SK-MEL-5 cells while prophalan-d was ineffective, suggesting that prolidase-specific activation to the parent drug may be essential for cytotoxic action. Thus, melphalan prodrugs such as prophalan-l that are cleavable by prolidase offer the potential for enhanced selectivity by facilitating cytotoxic activity only in cells overexpressing prolidase.

Synthesis and in vivo biodisposition of [14C]-quaternary ammonium-melphalan conjugate, a potential cartilage-targeted alkylating drug.

For the purpose of developing more selective anticancer drugs that would concentrate in the malignant cartilaginous tumors (chondrosarcomas), and so improve therapeutic index through a reduction of side effects, a quaternary ammonium (QA) conjugate of melphalan was synthesized and labeled with (14)C by linking the QA moiety to nitrogen mustard via an amide bond. Comparative pharmacokinetic study of [(14)C]-melphalan and its [(14)C]-QA conjugate conducted on rats showed that the two compounds were principally excreted by the urinary way. The blood elimination of the QA conjugate was faster than that of the melphalan. In the other hand a higher rate of radioactivity derived of [(14)C]-MQA was found in feces. In the biodisposition for most organs, no striking differences were found between melphalan and its QA conjugate except for cartilages which exhibited more higher radioactivity level. Amounts of radioactivity derived from [(14)C]-QA conjugates measured in cartilaginous tissues until 1 h after injection demonstrate that the introduction of a QA moiety on melphalan allows the molecule to be carried selectively to cartilaginous tissues. As the [(14)C]-QA conjugate is radiolabeled on the chloroethyl alkylating moiety, levels of radioactivity measured in the cartilaginous tissues results from unchanged compound or metabolite having kept the active group.

Proline analogue of melphalan as a prolidase-convertible pro-drug in breast cancer MCF-7 cells.

Prolidase [E.C.3.4.13.9] is ubiquitously distributed cytosolic egzopeptidase that is known to cleave imido-bond of some low molecular weight compounds coupled to L-proline. Previously we have found that conjugation of antineoplastic drug--melphalan (Mel) with proline (pro) through imido-bond resulted in formation of a good substrate for purified prolidase. Cytosolic location of prolidase in neoplastic cells suggests that proline analogue of melphalan (Mel-pro) may serve as a prolidase convertable pro-drug. We have compared several aspects of pharmacologic actions of Mel and Mel-pro in breast cancer MCF-7 cells. It has been found that Mel-pro is more effectively transported into the MCF-7 cells, evokes higher cytotoxicity, lower antimitotic activity and collagen-inhibiting activity, compared to Mel. The results suggest that targeting of prolidase as a pro-drug-converting enzyme may serve as a potential strategy in pharmacotherapy of breast cancer.

Development and activities of a new melphalan prodrug designed for tumor-selective activation.

The synthesis of C-Mel, a cephalosporin carbamate derivative of the clinically used alkylating agent melphalan, is described. C-Mel was designed as an anticancer nitrogen mustard prodrug that releases melphalan upon tumor-specific activation by targeted beta-lactamase (bL). The Km and kcat values for bL hydrolysis of C-Mel were 218 microM and 980 s(-1), respectively. In vitro cytotoxicity assays with 3677 human melanoma cells demonstrated that C-Mel was 40-fold less toxic than melphalan and was activated in an immunologically specific manner by L49-sFv-bL, a recombinant fusion protein that binds to the melanotransferrin antigen on melanomas and on some carcinomas. L49-sFv-bL in combination with C-Mel led to regressions and cures of established subcutaneous 3677 tumors in nude mice. The effects were significantly greater than those of melphalan, which did not result in any long-term regressions in this tumor model. The therapeutic effects were comparable to those obtained in mice treated with the previously described L49-sFv-bL/7-(4-carboxybutanamido)-cephalosporin mustard (CCM) combination. However, C-Mel may be more attractive than CCM for clinical development since the released drug is clinically approved.

Modulation of melphalan resistance in glioma cells with a peripheral benzodiazepine receptor ligand-melphalan conjugate.

Peripheral benzodiazepine receptors (PBRs) are located on the outer membrane of mitochondria, and their density is increased in brain tumors. Thus, they may serve as a unique intracellular and selective target for antineoplastic agents. A PBR ligand-melphalan conjugate (PBR-MEL) was synthesized and evaluated for cytotoxicity and affinity for PBRs. PBR-MEL (9) (i.e., 670 amu) was synthesized by coupling of two key intermediates: 4-[bis(2-chloroethyl)-amino]-L-phenylalanine ethyl ester trifluoroacetate (6) and 1-(3'-carboxylpropyl)-7-chloro-1,3- dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one (8). On the basis of receptor-binding displacement assays in rat brain and glioma cells, 9 had appreciable binding affinity and displaced a prototypical PBR ligand, Ro 5-4864, with IC50 values between 289 and 390 nM. 9 displayed differential cytotoxicity to a variety of rat and human brain tumor cell lines. In some of the cell lines tested including rat and human melphalan-resistant cell lines, 9 demonstrated appreciable cytotoxicity with IC50 values in the micromolar range, lower than that of melphalan alone. The enhanced activity of 9 may reflect increased membrane permeability, increased intracellular retention, or modulation of melphalan's mechanisms of resistance. The combined data support additional studies to determine how 9 may modulate melphalan resistance, its mechanisms of action, and if target selectivity can be achieved in in vivo glioma models.

Derivatives of melphalan designed to enhance drug accumulation in cancer cells.

The objective of this study was to develop chemical strategies to improve the uptake and accumulation of melphalan (L-Mel and D-Mel), a cytotoxic agent, into cancer cells. Dipeptides synthesized from L- (or D-) Mel and L-glutamic acid (L-Glu) or L-valine (L-Val) and their methyl or ethyl esters (all compounds were trifluoroacetic acid salts) were evaluated for cytotoxicity and cellular uptake using Caco-2 cells, a human colon carcinoma cell line, and RT-2 cells, a rat brain glioma cell line. Treatment of Caco-2 cells with L-Mel or D-Mel (0.5 mg/ml equivalent of melphalan) for 48 h resulted in approximately 50% cell survival. Treatment of the Caco-2 cells with dipeptide derivatives of L-Mel (or D-Mel) (11c-d, 12c-d and 13) caused similar cytotoxicity effects (approximately 50-70% of cell survival). When the cytotoxicities of the esters of L-Mel, D-Mel and their dipeptide derivatives (11a-b, 12a-b and 14) in Caco-2 cells were determined, less than 10% cell survival was observed. Similar results were observed in RT-2 cells. When the cellular uptake properties of these compounds were determined in Caco-2 cell monolayers, L-Glu-L-Mel (12c), L-Glu-D-Mel (12d), and L-Mel-L-Glu (11c) generated slightly lower intracellular levels of L-Mel or D-Mel than when the cell monolayer was treated with the amino acids (L-Mel or D-Mel). In Caco-2 cells treated with 11c, 12c or 12d, low levels of the dipeptides were also detected. Caco-2 cell monolayers treated with D-Mel-L-Glu (11d) or D-Mel-L-Val (13) showed very low levels of the amino acids (L-Mel or D-Mel), but generally higher levels of the dipeptides. In contrast to the amino acids (L-Mel, D-Mel) or the dipeptide derivatives (11c-d, 12c-d and 13), the ester derivatives of the amino acids [L-Mel(OEt), D-Mel(OEt)] or the dipeptides (11a-b, 12a-b and 14) produced 5-20 times higher intracellular concentrations of potentially cytotoxic metabolites (e.g., L-Mel, D-Mel, Mel-containing dipeptides or Mel-containing dipeptide monoesters). L-Mel(OEt), D-Mel(OEt), L-Glu(OEt)-L-Mel(OEt) (12a), L-Glu(OEt)-D-Mel(OEt) (12b), and L-Mel-L-Glu(OEt)2 (11a) accumulated mainly as either L-Mel or D-Mel, and the percentages of L-Mel or D-Mel were 99%, 99%, 90%, 75% and 98% of the total intracellular concentration of potentially cytotoxic agents, respectively. D-Mel-L-Glu(OEt)2 (11b) accumulated as its monoester (> 95%) and D-Mel-L-Val(OMe) (14) accumulated as its dipeptide metabolite (> 98%). Inclusion of Gly-Pro, carnosine, L-Phe or L-Glu did not inhibit uptake of the dipeptide derivatives of L-Mel (or D-Mel) or their esters. These results suggest that the cellular uptake of the dipeptide derivatives of melphalan and their esters is probably via passive diffusion rather than being facilitated by an amino acid transporter or a di/tripeptide transporter. The higher intracellular levels of cytotoxic agents generated from the ester derivatives of the amino acids and the dipeptides are probably due to their higher lipophilicity and the overall neutral charge of the esters and subsequent intracellular formation of the more polar amino acids (L- or D-Mel) and/or Mel-containing dipeptides. Finally, these studies suggest that dipeptides of D-Mel [11b, 11d, 13] have inherent cytotoxicity properties.

Electrospray mass spectrometric analysis of new alpha-MSH analogues carrying nitrogen mustard at their amino terminus.

Two different chlorinated drugs, chlorambucil and melphalan, have been linked to the carrier alpha-melanocyte-stimulating hormone at the end of the solid-phase peptide synthesis of the hormone. The [Nle4, D-Phe7] and the [Nle4, L-Phe7] derivatives of the hormone have both been used. It was found by electrospray mass spectrometric analysis that the products undergo hydrolysis of the chlorinated moiety of the drugs, most likely due to the acidic conditions necessary for cleavage of the peptide from the resin. Only the melphalan-O(ethyl)-N(succinyl)-derivative of alpha-melanocyte-stimulating hormone [Nle4, L-Phe7] did not show a bis-hydroxylated form. It was proven by blank experiments with commercially available melphalan that this mustard occurs for some 45%-50% in the mono-hydroxylated form, which is known to be pharmacologically poorly active.