1-[4-(2-Dimethylaminoethoxy)phenylcarbonyl]-3,5-Bis(3,4,5-Trimethoxybenzylidene)- 4-Piperidone Hydrochloride and Related Compounds: Potent Cytotoxins Demonstrate Greater Toxicity to Neoplasms than Non- Malignant Cells.

BACKGROUND: The incidence of cancer has been increasing worldwide. Unfortunately, the drugs used in cancer chemotherapy are toxic to both neoplasms and normal tissues, while many available medications have low potencies. Conjugated α,ß-unsaturated ketones differ structurally from contemporary anticancer medications , some of which have noteworthy antineoplastic properties. OBJECTIVES: This study aimed to design and synthesize highly potent cytotoxins with far greater toxicity to neoplasms than to non-malignant cells. METHODS: A series of N-acyl-3,5-bis(benzylidene)-4-piperidone hydrochlorides 4a-n were prepared and evaluated against Ca9-22, HSC-2, HSC-3, and HSC-4 squamous cell carcinomas as well as against HGF, HPLF, and HPC non-malignant cells. QSAR and western blot analyses were performed. RESULTS: The majority of compounds display submicromolar CC50 values towards the neoplasms; the figures for some of the compounds are below 10-7 M. In general, 4a-n have much lower CC50 values than those of melphalan, 5-fluorouracil, and methotrexate, while some compounds are equitoxic with doxorubicin. The compounds are far less toxic to the non-malignant cells, giving rise to substantial selectivity index (SI) figures. A QSAR study revealed that both potency and the SI data were controlled to a large extent by the electronic properties of the substituents in the arylidene aryl rings. Two representative compounds, 4f and 4g, caused apoptosis in HSC-2 cells. CONCLUSION: The compounds in series 4 are potent cytotoxins displaying tumor-selective toxicity. In particular, 4g with an average CC50 value of 0.04 µM towards four malignant cell lines and a selectivity index of 46.3 is clearly a lead molecule that should be further evaluated.

Structure⁻Activity Relationship of Piplartine and Synthetic Analogues against Schistosoma mansoni and Cytotoxicity to Mammalian Cells.

Schistosomiasis, caused by helminth flatworms of the genus Schistosoma, is an infectious disease mainly associated with poverty that affects millions of people worldwide. Since treatment for this disease relies only on the use of praziquantel, there is an urgent need to identify new antischistosomal drugs. Piplartine is an amide alkaloid found in several Piper species (Piperaceae) that exhibits antischistosomal properties. The aim of this study was to evaluate the structure­function relationship between piplartine and its five synthetic analogues (19A, 1G, 1M, 14B and 6B) against Schistosoma mansoni adult worms, as well as its cytotoxicity to mammalian cells using murine fibroblast (NIH-3T3) and BALB/cN macrophage (J774A.1) cell lines. In addition, density functional theory calculations and in silico analysis were used to predict physicochemical and toxicity parameters. Bioassays revealed that piplartine is active against S. mansoni at low concentrations (5⁻10 µM), but its analogues did not. In contrast, based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, piplartine exhibited toxicity in mammalian cells at 785 µM, while its analogues 19A and 6B did not reduce cell viability at the same concentrations. This study demonstrated that piplartine analogues showed less activity against S. mansoni but presented lower toxicity than piplartine.

Synthesis and Evaluation of Anti-inflammatory N-Substituted 3,5-Bis(2-(trifluoromethyl)benzylidene)piperidin-4-ones.

A total of 24 N-substituted 3,5-bis(2-(trifluoromethyl)benzylidene)piperidin-4-one derivatives were synthesized via aldol condensation, and their anti-inflammatory activities were evaluated. These compounds were found to have no significant cytotoxicity against mouse bone marrow cells in vitro. However, some compounds, such as c6 (N-(3-methylbenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)piperidin-4-one) and c10 (N-(2-chlorobenzoyl)-3,5-bis-(2-(trifluoromethyl)benzylidene)piperidin-4-one), displayed potent anti-inflammatory activity by inhibiting lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), IL-1ß, prostaglandin E2 (PGE2), and nitric oxide (NO) production in RAW 264.7 cells. Treatment with c6 or c10 at 2.5 or 10 mg kg-1 significantly decreased the paw edema induced by carrageenan in rats, and the anti-inflammatory effects of these compounds were found to be better than those of celecoxib or indomethacin as well as their parent compound C66 (2,6-bis-(2-(trifluoromethyl)benzylidene)cyclohexanone). Pharmacokinetic analysis indicated that c6 has better bioavailability than curcumin. Therefore, these compounds may be valuable leads for the development of new anti-inflammatory drugs.

Nanoemulsion formulations for anti-cancer agent piplartine--Characterization, toxicological, pharmacokinetics and efficacy studies.

Piplartine (PL) is an alkaloid found in black-pepper and known for its anticancer activity, however, due to poor solubility and lack of proper formulation, its use for oral administration is a challenge. The objective of this study was to formulate PL into nanoemulsion drug delivery system for oral delivery and thereafter evaluate toxicity, pharmacokinetics and therapeutic efficacy. Optimized nanoemulsions were formulated by self-emulsification as well as by homogenization-sonication method. Two nanoemulsions enhanced the solubility of PL with low polydispersity index and high stability. Both PL loaded nanoemulsions exhibited enhanced dissolution, cellular permeability and cytotoxic effects as compared to pure PL. Formulation of PL into nanoemulsions did not obstruct its cellular uptake in cancer cells. Blank or PL loaded nanoemulsions did not exhibited toxicity in mice upon daily oral administration for 60 days. Pharmacokinetics of PL followed a two-compartment model after intravenous administration. PL loaded nanoemulsions showed 1.5-fold increase in oral bioavailability as compared to free PL. Finally, PL loaded nanoemulsions showed marked anti-tumor activity at a dose of 10mg/kg in melanoma tumor bearing mice. In conclusion, for the first time we have developed a stable nanoemulsion delivery system for oral administration of PL, which enhanced its solubility, oral bioavailability and anti-tumor efficacy.

HO-3867, a curcumin analog, sensitizes cisplatin-resistant ovarian carcinoma, leading to therapeutic synergy through STAT3 inhibition.

Cisplatin resistance is a major obstacle in the treatment of ovarian cancer. Drug combinations with synergistic or complementary functions are a promising strategy to overcome this issue. We studied the anticancer efficacy of a novel compound, HO-3867, used in combination with cisplatin against chemotherapy-resistant ovarian cancer. A2780R cells, a cisplatin-resistant human ovarian cancer cell line, were exposed to 1, 5, or 10 uM of HO-3867 alone or in combination with cisplatin (10 ug/ml) for 24 hours. Cell viability (MTT), proliferation (BrdU), cell-cycle analysis (FACS), and protein expression (western blot) were used for in vitro studies. STAT3 overexpression was performed using transfected STAT3 cDNA. In vivo studies used cisplatin-resistant xenograft tumors grown in nude mice and treated with 100-ppm HO-3867 and weekly injections of 4-mg/kg cisplatin. HO-3867/cisplatin combination treatment significantly inhibited cisplatin-resistant cell proliferation in a concentration-dependent manner. The inhibition was associated with increased expression of p53 and p21, and decreased expression of cdk5 and cyclin D1. Apoptosis was induced by activation of Bax, cytochrome c release, and stimulated cleavage of caspase-9, caspase-3, and PARP. Overexpression of STAT3 decreased the HO-3867-induced apoptosis. The combination treatment significantly inhibited the growth of cisplatin-resistant xenograft tumors with significant downregulation of pSTAT3, and without apparent toxicity to healthy tissues. The combination treatment exhibited synergistic anticancer efficacy, which appears largely due to HO-3867-induced downregulation of pSTAT3. The results, combined with the previously-reported safety features of HO-3867, suggest the potential use of this compound as a safe and effective adjuvant for the treatment of ovarian cancer.

Bis[3,5-bis(benzylidene)-4-oxo-1-piperidinyl]amides: a novel class of potent cytotoxins.

The principal objective of this study was the examination of the theory of cytotoxic synergism. In this exploratory study, we tested the hypothesis that doubling the number of sites available for thiol alkylation in a series of candidate cytotoxins increases potency more than two-fold. This concept was verified in one-third of our comparisons using human Molt 4/C8 and CEM T-lymphocytes and murine L1210 cells. In addition, the significant potencies of various members of our compound series justified further studies. Molecular modeling revealed that relative locations of the amidic groups correlate with cytotoxicity. A potent cytotoxic compound, 1,2-bis(3,5-dibenzylidene-4-oxo-piperidin-1-yl)ethane-1,2-dione (1a) inhibited the growth of a large number of human tumor cell lines and displayed greater toxicity toward certain non-adherent cells than toward adherent neoplasms or fibroblasts. The mode of action of 1a includes induction of apoptosis and necrosis.

3,5-Bis(benzylidene)-4-oxo-1-phosphonopiperidines and related diethyl esters: Potent cytotoxins with multi-drug-resistance reverting properties.

A series of 3,5-bis(benzylidene)-4-piperidones 3 were converted into the corresponding 3,5-bis(benzylidene)-1-phosphono-4-piperidones 5 via diethyl esters 4. The analogues in series 4 and 5 displayed marked growth inhibitory properties toward human Molt 4/C8 and CEM T-lymphocytes as well as murine leukemia L1210 cells. In general, the N-phosphono compounds 5, which are more hydrophilic than the analogues in series 3 and 4, were the most potent cluster of cytotoxins, and, in particular, 3,5-bis-(2-nitrobenzylidene)-1-phosphono-4-piperidone 5 g had an average IC(50) value of 34 nM toward the two T-lymphocyte cell lines. Four of the compounds displayed potent cytotoxicity toward a panel of nearly 60 human tumor cell lines, and nanomolar IC(50) values were observed in a number of cases. The mode of action of 5 g includes the induction of apoptosis and inhibition of cellular respiration. Most of the members of series 4 as well as several analogues in series 5 are potent multi-drug resistance (MDR) reverting compounds. Various correlations were noted between certain molecular features of series 4 and 5 and cytotoxic properties, affording some guidelines in expanding this study.

Piplartine induces genotoxicity in eukaryotic but not in prokaryotic model systems.

Piplartine {5,6-dihydro-1-[(2E)-1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propen-1-yl]-2(1H)-pyridinone} is an alkamide present in Piper species that exhibits promising anticancer properties. It was previously shown that piplartine is mutagenic in yeast and cultured mammalian cells. This study was performed to increase the knowledge on the mutagenic potential of piplartine using the Salmonella/microsome assay, V79 cell micronucleus and chromosome aberration assays, and mouse bone-marrow micronucleus tests. Piplartine was isolated from the roots of Piper tuberculatum. This extracted compound was unable to induce a mutagenic response in any Salmonella typhimurium strain either in the presence or absence of metabolic activation. Piplartine showed mutagenic effects in V79 cells, as there was an increased frequency of aberrant cells and micronuclei formation. In addition, piplartine administered at 50mg/kg did not induce micronucleus formation in vivo, but a dose of 100mg/kg induced an increase in the levels of micronucleus polychromatic erythrocytes (MNPCEs). Overall, these results provide further support that piplartine induces in vivo and in vitro mutagenicity in eukaryotic models.

Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells.

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary -- or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.

Cytotoxic 3,5-bis(benzylidene)piperidin-4-ones and N-acyl analogs displaying selective toxicity for malignant cells.

A series of 3,5-bis(benzylidene)piperidin-4-ones 1, 1-acryloyl-3,5-bis(benzylidene)piperidin-4-ones 2 and adducts of 2 with sodium 2-mercaptoethanesulfonate (mesna), namely series 3, were prepared as candidate cytotoxic agents. These compounds were examined against neoplastic HSC-2, HSC-4 and HL-60 cells as well as HGF, HPC and HPLF normal cell lines and many of the compounds displayed selective toxicity for malignant cells. The CC50 values of the analogs in series 2 towards the cancer cell lines were mainly submicromolar. The relative potencies, selectivity and logP values were in the order of 2>1>3. The sulfonic acid group of a representative compound in series 3 was replaced by a thiol function to produce 4 leading to substantial increases in cytotoxic potencies and hydrophobicity indicating that the presence of a hydrophilic sulfonic acid group was disadvantageous in terms of potency. Molecular modeling suggested that the superior cytotoxicity of various members of series 1-3 over an acyclic analog 5 may have been due to the greater torsion angles theta1 and theta2 created between the arylidene aryl rings and the adjacent olefinic groups in series 1-3.

CoMFA and CoMSIA studies on 1,3-bis(benzylidene)-3,4-dihydro-1H-naphthalen-2-one, 2,6-bis(benzylidene)cyclohexanone, and 3,5-bis(benzylidene)-4-piperidone series of cytotoxic compounds.

A number of 1,3-bis(benzylidene)-3,4-dihydro-1H-naphthalen-2-ones, 2,6-bis(benzylidene)cyclohexanones, and 3,5-bis(benzylidene)-4-piperidones possess significant potencies toward L1210, Molt 4/C8, and CEM cell lines. The objective of the current 3D QSAR study is to discover some of the structural parameters which govern cytotoxic potencies. The CoMFA models with steric and electrostatic fields provided satisfactory statistical data [(r2cv = 0.485, r2ncv = 0.834, r2pred = 0.591), (r2cv = 0.532, r2ncv = 0.850, r2pred = 0.729), and (r2cv = 0.561, r2ncv = 0.864, r2pred = 0.666)] in regard to the cytotoxic potencies observed toward L1210, Molt 4/C8, and CEM cell lines, respectively. The CoMSIA model with steric, electrostatic, hydrophobic, and H-bond donor fields exhibited r2cv = 0.513, r2ncv = 0.833, and r2pred = 0.562 for cytotoxic activity toward L1210 cells, while the best CoMSIA models were obtained by a combination of steric, electrostatic, and hydrophobic fields which yielded statistically significant data [(r2cv = 0.531, r2ncv = 0.828, r2pred = 0.652) and (r2cv = 0.560, r2ncv = 0.841, r2pred = 0.729)] to explain the cytotoxicity toward Molt 4/C8 and CEM cells, respectively. The information obtained from the CoMFA and CoMSIA 3D contour maps can be used in the design of more potent cytotoxins.

E,E,E-1-(4-Arylamino-4-oxo-2-butenoyl)-3,5-bis(arylidene)-4-piperidones: a topographical study of some novel potent cytotoxins.

A series of E,E,E-3,5-bis(arylidene)-1-(4-arylamino-4-oxo-2-butenoyl)-4-piperidones 4 (phenylidene) and 5 (4-nitrophenylidene) were prepared in order to explore the structural features of the N-acyl group which affects the cytotoxic potency. Evaluation toward human Molt 4/C8 and CEM T-lymphocytes revealed that many of the IC(50) figures were submicromolar and lower than melphalan. Marked inhibitory potencies toward murine leukemia L1210 cells were also noted. When evaluated against a panel of human tumor cell lines, three representative compounds in series 4 displayed selective toxicity to leukemia and colon cancer cell lines and were significantly more potent than the reference drug melphalan. Molecular modeling of representative compounds in both series 4 and the analogs, in which the configuration of the olefinic double bond was changed from E to Z (series 3), revealed that the torsion angles of the arylidene aryl rings and locations of the terminal arylaminocarbonyl groups may have contributed to the greater cytotoxic properties displayed in 3. Compounds 4c (3,4-dichlorophenylamino), d (4-methylphenylamino) and 5c (3,4-dichlorophenylamino), d (4-methylphenylamino) inhibited the activity of human N-myristoyltransferase by approximately 50% at concentrations of 50-100 microM. The compounds in series 4 and 5 were well tolerated in a short-term toxicity study in mice.

In vivo growth-inhibition of Sarcoma 180 by piplartine and piperine, two alkaloid amides from Piper.

Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} and piperine {1-5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine} are alkaloid amides isolated from Piper. Both have been reported to show cytotoxic activity towards several tumor cell lines. In the present study, the in vivo antitumor activity of these compounds was evaluated in 60 female Swiss mice (N = 10 per group) transplanted with Sarcoma 180. Histopathological and morphological analyses of the tumor and the organs, including liver, spleen, and kidney, were performed in order to evaluate the toxicological aspects of the treatment with these amides. Administration of piplartine or piperine (50 or 100 mg kg(-1) day(-1) intraperitoneally for 7 days starting 1 day after inoculation) inhibited solid tumor development in mice transplanted with Sarcoma 180 cells. The inhibition rates were 28.7 and 52.3% for piplartine and 55.1 and 56.8% for piperine, after 7 days of treatment, at the lower and higher doses, respectively. The antitumor activity of piplartine was related to inhibition of the tumor proliferation rate, as observed by reduction of Ki67 staining, a nuclear antigen associated with G1, S, G2, and M cell cycle phases, in tumors from treated animals. However, piperine did not inhibit cell proliferation as observed in Ki67 immunohistochemical analysis. Histopathological analysis of liver and kidney showed that both organs were reversibly affected by piplartine and piperine treatment, but in a different way. Piperine was more toxic to the liver, leading to ballooning degeneration of hepatocytes, accompanied by microvesicular steatosis in some areas, than piplartine which, in turn, was more toxic to the kidney, leading to discrete hydropic changes of the proximal tubular and glomerular epithelium and tubular hemorrhage in treated animals.

In vivo growth-inhibition of Sarcoma 180 by piplartine and piperine, two alkaloid amides from Piper

Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} and piperine {1-5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine} are alkaloid amides isolated from Piper. Both have been reported to show cytotoxic activity towards several tumor cell lines. In the present study, the in vivo antitumor activity of these compounds was evaluated in 60 female Swiss mice (N = 10 per group) transplanted with Sarcoma 180. Histopathological and morphological analyses of the tumor and the organs, including liver, spleen, and kidney, were performed in order to evaluate the toxicological aspects of the treatment with these amides. Administration of piplartine or piperine (50 or 100 mg kg-1 day-1 intraperitoneally for 7 days starting 1 day after inoculation) inhibited solid tumor development in mice transplanted with Sarcoma 180 cells. The inhibition rates were 28.7 and 52.3 percent for piplartine and 55.1 and 56.8 percent for piperine, after 7 days of treatment, at the lower and higher doses, respectively. The antitumor activity of piplartine was related to inhibition of the tumor proliferation rate, as observed by reduction of Ki67 staining, a nuclear antigen associated with G1, S, G2, and M cell cycle phases, in tumors from treated animals. However, piperine did not inhibit cell proliferation as observed in Ki67 immunohistochemical analysis. Histopathological analysis of liver and kidney showed that both organs were reversibly affected by piplartine and piperine treatment, but in a different way. Piperine was more toxic to the liver, leading to ballooning degeneration of hepatocytes, accompanied by microvesicular steatosis in some areas, than piplartine which, in turn, was more toxic to the kidney, leading to discrete hydropic changes of the proximal tubular and glomerular epithelium and tubular hemorrhage in treated animals.

Antiproliferative effects of two amides, piperine and piplartine, from Piper species.

The present work evaluated the cytotoxicity of piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-trans-2-propenyl]-2(1H)pyridinone} and piperine {1-[5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine}, components obtained from Piper species. The substances were tested for their cytotoxicity on the brine shrimp lethality assay, sea urchin eggs development, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using tumor cell lines and lytic activity on mouse erythrocytes. Piperine showed higher toxicity in brine shrimp (DL50 = 2.8 +/- 0.3 microg/ml) than piplartine (DL50 = 32.3 +/- 3.4 microg/ml). Both piplartine and piperine inhibited the sea urchin eggs development during all phases examined, first and third cleavage and blastulae, but in this assay piplartine was more potent than piperine. In the MTT assay, piplartine was the most active with IC50 values in the range of 0.7 to 1.7 microg/ml. None of the tested substances induced hemolysis of mouse erythrocytes, suggesting that the cytotoxicity of piplartine and piperine was not related to membrane damage.

Synthesis of curcumin analogues as potential antioxidant, cancer chemopreventive agents.

New series of 3, 5-bis(substituted benzylidene)-4-piperidones, 2, 7-bis(substituted benzylidene)cycloheptanones, 1, 5-bis(substituted phenyl)-1, 4-pentadien-3-ones, 1, 7-bis(substituted phenyl)-1, 6-heptadien-3, 5-diones, 1, 1-bis(substituted cinnamoyl)-cyclopentanes, and 1, 1-bis(substituted cinnamoyl)cyclohexanes have been synthesized and tested for their antioxidant activity. Among the tested compounds, compounds II(4), II(9) II(10), II(11), V(1), and V(4) exhibited higher free radical scavenger activity with % inhibition values of 90.71, 91.24, 96.91, 94.26, 99.23, and 99.85%, respectively. Moreover, compound V(1) is the safest member toward peripheral multinuclear neutrophils (PMNs) with a % viability value of 91%. Detailed synthesis, spectroscopic, and biological data are reported.

3,5-bis(phenylmethylene)-1-(N-arylmaleamoyl)-4-piperidones: a novel group of cytotoxic agents.

A series of novel 3,5-bis(phenylmethylene)-1-(N-arylmaleamoyl)-4-piperidones 3 have been synthesized which displayed potent cytotoxicity towards human Molt 4/C8 and CEM T-lymphocytes as well as murine P388 and L1210 leukemic cells. In contrast, the related N-arylmaleamic acids 4 possessed little or no cytotoxicity in these four screens. Molecular modeling revealed certain interplanar and bond angles and interatomic distances which were perceived to contribute to the observed bioactivity as well as providing suggestions for future structural modifications of the piperidones 3. Evaluation of representative compounds in series 3 and 4 on the activity of human N-myristoyltransferase revealed that, at the maximum concentration utilized, namely 250 microM, only weak inhibiting properties were displayed by some of the compounds in series 4. Various members of series 3 and 4 were well tolerated in mice.

Cytotoxic N-[4-(3-aryl-3-oxo-1-propenyl)phenylcarbonyl]-3,5-bis(phenylmethylene)-4-piperidones and related compounds.

A series of 4-carboxychalcones 1 were prepared and coupled to 3,5-bis(phenylmethylene)-4-piperidone (2) giving rise to a novel series of N-[4-(3-aryl-3-oxo-1-propenyl)phenylcarbonyl]-3,5-bis(phenylmethylene)-4-piperidones (3). Molecular simplification of the amides 3 led to the formation of the corresponding N-(3-aryl-1-oxo-2-propenyl)-3,5-bis(phenylmethylene)-4-piperidones (4). A cytotoxic evaluation of the compounds in series 1-4 utilized murine P388 and L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes. In general, the compounds displayed significant toxicity; the IC(50) values of 54% of the enones were less than 10 microM when all four screens were considered and less than 1 microM for all members of series 3 in the P388 assay. Various correlations were established between the potencies of the compounds in series 1, 3 and 4 and the Hammett sigma, Hansch pi and molecular refractivity constants of the aryl substituents. Several torsion angles and interatomic distances of five representative compounds in series 3 and 4 were determined by X-ray crystallography, some of which contributed to the observed bioactivity. The marked cytotoxicity and lack of murine toxicity of most of the compounds described in this study, as well as their selective toxicity towards different tumour cell lines, revealed that development of the enones 2-4 as novel candidate antineoplastic agents should be pursued.

Cytotoxic evaluation of some N-acyl and N-acyloxy analogues of 3,5-bis(arylidene)-4-piperidones.

The preparation of thirteen N-acyl and N-acyloxy analogues of two 3,5-bis(arylidene)-4-piperidones were prepared as potential prodrugs of the parent ketones. Approximately half of the compounds demonstrated antileukemic properties when evaluated against murine L1210 lymphoid leukemia cells. Three of the derivatives were more active than or equipotent with the reference drug melphalan. All of the compounds were examined against approximately 55 human tumours representing eight different neoplastic diseases. Not only were most of the compounds more cytotoxic than melphalan but 60% of the derivatives displayed selective toxicity to leukemia. A stability study of six of the candidate prodrugs using 1H NMR spectroscopy revealed that while some decomposition in solution occurred, the parent amine was not liberated.

3-phenylacetylamino-2,6-piperidinedione inhibition of rat Nb2 lymphoma cell mitogenesis.

3-Phenylacetylamino-2,6-piperidinedione (A10), an amino acid analog, has been reported to possess antineoplastic activity against certain neoplastic tissues. The antimitogenic properties of A10 were studied by determining its effect on prolactin (PRL)- and interleukin 2 (IL-2)-stimulated mitogenic responses in the rat Nb2 lymphoma cell line. The addition of A10 (1-12 mM) to PRL (0.4 ng/ml)-stimulated cells inhibited growth in a dose-dependent manner. DNA synthesis patterns studied by thymidine incorporation demonstrated that A10 was significantly inhibitory (25% at 20 hr; 50% at 40 hr, P less than 0.01). IL-2 stimulation of mitogenesis was also sensitive to A10 inhibition. The inhibition of PRL stimulated mitogenesis was reversible when A10 was removed after 24 hr of culture and A10 showed no toxicity in a chromium release assay. These data suggest that A10 effects may be cytostatic, rather than cytotoxic.