Since our newly synthesized potent 5-indolyl derivative, (2-(1 H-Indol-5-yl) thiazol-4-yl) 3, 4, 5-trimethoxyphenyl methanone (LY293), to treat resistant melanoma was hydrophobic, our objective was to synthesize a biodegradable copolymer for formulating this drug into nanoparticles and to determine its anticancer activity and mechanism of action.
Methoxy poly (ethylene glycol)-b-poly (carbonate-colactide) [mPEG-b-P (CB-co-LA)] was synthesized for formulating LY293 into nanoparticles by o/w emulsification and stabilization by solvent evaporation. Particle size, drug release profile, in vitro efficacy in multiple melanoma cells, and mechanism of action of drug-loaded nanoparticles were determined.
LY293-loaded nanoparticles with 170 nm mean size and 2.2 and 4.16% drug loading efficiently inhibited proliferation of A375 and B16F10 cells with IC50 of 12.5 nM and 25 nM, respectively. LY293 circumvented multidrug resistance and inhibited proliferation of Pgp overexpressing MDA-MB435/LCC6 MDR1 melanoma cells. Upon treatment with LY293-loaded nanoparticles, A375 cells underwent cell cycle arrest in G2/M phase and apoptotic cell death. Immunofluorescence images showed inhibition of tubulin polymerization after treatment with LY293.
LY293-loaded mPEG-b-P (CB-co-LA) nanoparticles showed excellent efficacy and induced apoptosis in melanoma cells. These polyester/polycarbonate-based nanoparticles provided an excellent platform to deliver different poorly soluble drugs to melanoma.
LY293; melanoma; polymeric nanoparticles; tubulin polymerization
To evaluate the efficacy and oral activity of two promising indoles, (2-(1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone [compound II] and (2-(1H-indol-5-ylamino)-thiazol-4-yl)(3,4,5-trimethoxyphenyl)methanone [compound IAT], in paclitaxel- and docetaxel-resistant tumor models in vitro and in vivo.
The in vitro drug-like properties, including potency, solubility, metabolic stability, and drug-drug interactions were examined for our two active compounds. An in vivo pharmacokinetic study and antitumor efficacy study were also completed to compare their efficacy with docetaxel.
Both compounds bound to the colchicine-binding site on tubulin, and inhibited tubulin polymerization, resulting in highly potent cytotoxic activity in vitro. While the potency of paclitaxel and docetaxel was compromised in a multidrug-resistant cell line that overexpresses P-glycoprotein, the potency of compounds II and IATwas maintained. Both compounds had favorable drug-like properties, and acceptable oral bioavailability (21–50%) in mice, rats, and dogs. Tumor growth inhibition of greater than 100% was achieved when immunodeficient mice with rapidly growing paclitaxel-resistant prostate cancer cells were treated orally at doses of 3–30 mg/kg of II or IAT.
These studies highlight the potent and broad anticancer activity of two orally bioavailable compounds, offering significant pharmacologic advantage over existing drugs of this class for multidrug resistant or taxane-refractory cancers.
paclitaxel resistant cancer; P-glycoprotein; pharmacokinetics; tubulin; xenograft
Tubulin dynamics is a promising target for new chemotherapeutic agents. The colchicine binding site is one of the most important pockets for potential tubulin polymerization destabilizers. Colchicine binding site inhibitors (CBSI) exert their biological effects by inhibiting tubulin assembly and suppressing microtubule formation. A large number of molecules interacting with the colchicine binding site have been designed and synthesized with significant structural diversity. CBSIs have been modified as to chemical structure as well as pharmacokinetic properties, and tested in order to find a highly potent, low toxicity agent for treatment of cancers. CBSIs are believed to act by a common mechanism via binding to the colchicine site on tubulin. The present review is a synopsis of compounds that have been reported in the past decade that have provided an increase in our understanding of the actions of CBSIs.
antimitotic; cancer; colchicine; multidrug resistance; tubulin polymerization inhibitor
Novel ABI–III compounds were designed and synthesized based on our previously reported ABI-I and ABI–II analogs. ABI–III compounds are highly potent against a panel of melanoma and prostate cancer cell lines, with the best compound having an average IC50 value of 3.8 nM. They are not substrate of Pgp and thus may effectively overcome Pgp mediated multidrug resistance. ABI–III analogs maintain their mechanisms of action by inhibition of tubulin polymerization.
Melanoma; prostate cancer; multidrug resistance; tubulin polymerization inhibitor; antiproliferative activity
To determine whether Compound 49b, a novel beta-adrenergic receptor agonist, can prevent increased inflammation and apoptosis in mice after exposure to ocular blast.
Eyes of C57/BL6 mice were exposed to a blast of air from a paintball gun at 26 psi (≈0.18 MPa). Eyes were collected 4 hours, 24 hours, and 72 hours after blast exposure. In a subset of mice, Compound 49b eyedrops (1 mM) were applied within 4 hours, 24 hours, or 72 hours of the blast. Three days after blast exposure, all mice were sacrificed. One eye was used to measure levels of retinal proteins (TNFα, IL-1β, Bax, BcL-xL, caspase 3, and cytochrome C). The other eye was used for TUNEL labeling of apoptotic cells, which were co-labeled with NeuN to stain for retinal ganglion cells.
We found that ocular exposure to 26 psi air pressure led to a significant increase in levels of apoptotic and inflammatory mediators within 4 hours, which lasted throughout the period investigated. When Compound 49b was applied within 4 hours or 24 hours of blast injury, levels of apoptotic and inflammatory mediators were significantly reduced. Application of Compound 49b within 72 hours of blast injury reduced levels of inflammatory mediators, but not to untreated levels.
Ocular blast injury produces a significant increase in levels of key inflammatory and apoptotic markers in the retina as early as 4 hours after blast exposure. These levels are significantly reduced if a beta-adrenergic receptor agonist is applied within 24 hours of blast exposure. Data suggest that local application of beta-adrenergic receptor agonists may be beneficial to reduce inflammation and apoptosis.
Apoptosis; Beta-adrenergic receptor agonists; Cytokines
As a continuation of our efforts to discover and develop small molecules as anticancer agents, we identified GRI-394837 as an initial hit from similarity search on RGD and its analogs. Based on GRI-394837, we designed and synthesized a focused set of novel chromenes (4a–e) in a single step using microwave method. All five compounds showed activity in the nanomolar range (IC50: 7.4–640 nM) in two melanoma, three prostate and four glioma cancer cell lines. The chromene 4e is active against all the cell lines and particularly against the A172 human glioma cell line (IC50: 7.4 nM). Interestingly, in vitro tubulin polymerization assay shows 4e to be a weak tubulin polymerization inhibitor but it shows very strong cytotoxicity in cellular assays, therefore there must be additional unknown mechanism(s) for the anticancer activity. Additionally, the strong antiproliferative activity was verified by one of the selected chromene (4a) by the NCI 60 cell line screen. These results strongly suggest that the novel chromenes could be further developed as a potential therapeutic agent for a variety of aggressive cancers.
Chromenes; RGD; Melanoma; Glioma; Prostate cancer; Tubulin polymerization inhibitors
Non-naturally occurring 20R epimer of 20-hydroxyvitamin D3 is synthesized based on chemical design and hypothesis. The 20R isomer is separated from semi-preparative HPLC and its structure is characterized. The comparison of 20R isomer to its 20S counterpart in biological evaluation demonstrates they have different behaviours in both antiproliferative and metabolic studies.
20R-hydroxyvitamin D3; 20S-hydroxyvitamin D3; chemical synthesis; NMR; antiproliferative activity; metabolism; CYP11A1; CYP27B1
LPA (lysophosphatidic acid, 1-acyl-2-hydroxy-sn-glycero-3-phosphate), is a growth factor-like lipid mediator that regulates many cellular functions, many of which are unique to malignantly transformed cells. The simple chemical structure of LPA and its profound effects in cancer cells has attracted the attention of the cancer therapeutics field and drives the development of therapeutics based on the LPA scaffold. In biological fluids, LPA is generated by ATX (autotaxin), a lysophospholipase D that cleaves the choline/serine headgroup from lysophosphatidylcholine and lysophosphatidylserine to generate LPA. In the present article, we review some of the key findings that make the ATX–LPA signalling axis an emerging target for cancer therapy.
autotaxin; cancer; drug discovery; lysophosphatidic acid (LPA); 4-pentadecylbenzylphosphonic acid (4-PBA); therapy
To determine whether Compound 49b, a novel PKA-activating drug, can prevent diabetic-like changes in the rat retina through increased insulin-like growth factor binding protein-3 (IGFBP-3) levels.
For the cell culture studies, we used both human retinal endothelial cells (REC) and retinal Müller cells in either 5 mM (normal) or 25 mM (high) glucose. Cells were treated with 50 nM Compound 49b alone of following treatment with protein kinase A (PKA) siRNA or IGFBP-3 siRNA. Western blotting and ELISA analyses were done to verify PKA and IGFBP-3 knockdown, as well as to measure apoptotic markers. For animal studies, we used streptozotocin-treated rats after 2 and 8 months of diabetes. Some rats were treated topically with 1 mM Compound 49b. Analyses were done for retinal thickness, cell numbers in the ganglion cell layer, pericyte ghosts, and numbers of degenerate capillaries, as well as electroretinogram and heart morphology.
Compound 49b requires active PKA and IGFBP-3 to prevent apoptosis of REC. Compound 49b significantly reduced the numbers of degenerate capillaries and pericyte ghosts, while preventing the decreased retinal thickness and loss of cells in the ganglion cell layer. Compound 49b maintained a normal electroretinogram, with no changes in blood pressure, intraocular pressure, or heart morphological changes.
Topical Compound 49b is able to prevent diabetic-like changes in the rat retina, without producing systemic changes. Compound 49b is able to prevent REC apoptosis through increasing IGFBP-3 levels, which are reduced in response to hyperglycemia.
We have developed a novel beta-adrenergic receptor agonist that prevents diabetic-like changes in the rat retina without systemic side effects. We have also determined the mechanism by which Compound 49b can prevent retinal endothelial cell apoptosis.
Background & Aims
We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. The objective of the present study was to evaluate the effect of octadecenyl thiophosphate (OTP), a novel rationally designed, metabolically stabilized LPA mimic, on radiation-induced apoptosis of intestinal epithelial cells in vitro and in vivo
The receptors and signaling pathways activated by OTP were examined in IEC-6 and RH7777 cell lines and wild-type and LPA1 and LPA2 knockout mice exposed to different apoptotic stimuli
OTP was more efficacious than LPA in reducing gamma irradiation–, camptothecin-, or tumor necrosis factor α/cycloheximide–induced apoptosis and caspase-3-8, and caspase-9 activity in the IEC-6 cell line. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA2 but not LPA1 and LPA3 transfectants. In C57BL/6 and LPA1 knockout mice exposed to 15 Gy gamma irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase-3–positive cells but was ineffective in LPA2 knockout mice. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but was without any effect in LPA2 knockout mice. Intraperitoneally administered OTP reduced death caused by lethal dose (LD)100/30 radiation by 50%.
Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA2 receptor subtype.
Prostate cancer cell migration is an essential event both in the progression of prostate cancer and in the steps leading to metastasis. We report here that lysophosphatidic acid (LPA), a potent bioactive phospholipid, induces prostate cancer PC3 cell migration via the activation of the LPA1 receptor, which is linked to a PTX-sensitive activation mechanism of the mitogen-activated protein kinases (MAPK). Our results demonstrate that parallel activation of ERK1/2 and p38, but not JNK, is responsible for LPA-stimulated PC3 cell migration. Furthermore, using small interfering RNA (siRNA) technology, and overexpressing dominant-negative mutants of p38 MAPK isotypes of α, β, γ and δ, we have identified that the activation of ERK2 (p42) and p38α, but not of ERK1 and the other isoforms of p38 MAPK, is required for LPA-induced migration. Our study provides the first evidence for a functional role of p42 and p38α in LPA-induced mammalian cell migration, and also demonstrates, for the first time, that the receptor LPA1 mediates prostate cancer cell migration. The results of the present study suggest that LPA, the receptor LPA1, ERK2 and p38α are important regulators for prostate cancer cell invasion and thus could play a significant role in the development of metastasis.
Lysophosphatidic acid; Receptors; Cell migration; Protein kinases and prostate cancer cells
A series of 4-substituted methoxylbenzoyl-aryl-thiazoles (SMART) have been discovered and synthesized as a result of structural modifications of the lead compound 2-arylthiazolidine-4-carboxylic acid amides (ATCAA). The antiproliferative activity of the SMART agents against melanoma and prostate cancer cells was improved from μM to low nM range compared with ATCAA series. The structure-activity relationship was discussed from modifications of “A”, “B” “C” rings and the linker. Preliminary mechanism of action studies indicated that these compounds exert their anticancer activity through inhibition of tubulin polymerization.
Thiazolidine; Thiazole; Melanoma; Prostate cancer; Antiproliferative activity; Structure-activity relationship; X-ray Crystal structure; Tubulin polymerization inhibitor
We previously reported the discovery of 2-aryl-4-benzoyl-imidazoles (ABI-I) as potent antiproliferative agents for melanoma. To further understand the structural requirements for the potency of ABI analogs, gain insight in the structure-activity relationships (SAR), and investigate metabolic stability for these compounds, we report extensive SAR studies on the ABI-I scaffold. Compared with the previous set of ABI-I analogs, the newly synthesized ABI-II analogs have lower potency in general, but some of the new analogs have comparable potency to the most active compounds in the previous set when tested in two melanoma and four prostate cancer cell lines. These SAR studies indicated that the antiproliferative activity was very sensitive to subtle changes in the ligand. Tested compounds 3ab and 8a are equally active against highly paclitaxel resistant cancer cell lines and their parental cell lines, indicating that drugs developed based on ABI-I analogs may have therapeutic advantages over paclitaxel in treating resistant tumors. Metabolic stability studies of compound 3ab revealed that N-methyl imidazole failed to extend stability as literature reported because de-methylation was found as the major metabolic pathway in rat and mouse liver microsomes. However, this sheds light on the possibility for many modifications on imidazole ring for further lead optimization since the modification on imidazole, such as compound 3ab, did not impact the potency.
We have synthesized 3β,21-dihydroxypregna-5,7-dien-20-one (21(OH) 7DHP) and used UVB radiation to induce its photoconversion to analogues of vitamin D (pD), lumisterol (pL) and tachysterol (pT). The number and character of the products and the dynamics of the process were dependent on the UVB dose. The main products: pD and pT compounds were characterized by UV absorption, MS and NMR spectroscopy after RP-HPLC chromatography. In addition, formation of multiple oxidized derivatives of the primary products was detected and one of these derivatives was characterized as oxidized 21-hydroxyisotachysterol compound (21(OH)oxy-piT). These newly synthesized compounds inhibited growth of human melanoma cells in a dose dependent manner, with greater or equal potency to calcitriol. 3β,21-Dihydroxy-9β,10α-pregna-5,7-dien-20-one (21(OH)pL) and 21(OH)oxy-piT had higher potency against pigmented melanoma cells, while the EC50 for compounds 21(OH)7DHP and (5Z,7E)-3β,21-dihydroxy-9,10-secopregna-5,7,10(19)-trien-20-one (21(OH)pD) were similar in both pigmented and non-pigmented cells. Moreover, 21(OH)7DHP and its derivatives inhibited proliferation of human epidermal HaCaT keratinocytes, albeit at a lower activity compared to melanoma cells. Importantly, 21(OH)7DHP derivatives strongly inhibited the colony formation of human melanoma cells with 21(OH)pD being the most potent. The potential mechanism of action of newly synthesized compounds was similar to that mediated by 1,25(OH)2D3 and involved ligand-induced translocation of vitamin D receptor into the nucleus. In summary, we have characterized for the first time products of UVB-induced conversion of 21(OH)7DHP and documented that these compounds have selective, inhibitory effects on melanoma cells.
secosteroids; UV radiation; lumisterol; vitamin D; oxidation; SLOS; skin; melanoma
Formation of microtubules is a dynamic process that involves polymerization and depolymerization of αβ-tubulin heterodimers. Drugs that enhance or inhibit tubulin polymerization can destroy this dynamic process, arresting cells in the G2/M phase of the cell cycle. Although drugs that target tubulin generally demonstrate cytotoxic potency in the sub-nanomolar range, resistance due to drug efflux is a common phenomenon among the antitubulin agents. We recently reported a class of 4-Substituted Methoxybenzoyl-Aryl-Thiazoles (SMART) that exhibited great in vitro potency and broad spectrum cellular cytotoxicity. Evaluation of the in vitro and in vivo anti-cancer activities of three SMART compounds, SMART-H (H), SMART-F (F) and SMART-OH (OH) with varying substituents at the 4-position of aryl ring, demonstrated that they bind potently to the colchicine binding site in tubulin, inhibit tubulin polymerization, arrest cancer cells in G2/M phase of the cell cycle, and induce their apoptosis.
The SMART compounds also equi-potently inhibit the growth of parental and MDR-over-expressing cells in vitro, indicating that they can overcome multidrug resistance. In vivo anti-tumor efficacy studies in human prostate (PC-3) and melanoma (A375) cancer xenograft models demonstrated that SMART-H and SMART-F treatments resulted in %T/C values ranging from 4–30%. In addition, in vivo SMART-H treatment for 21 days at the higher dose (15 mg/kg) failed to produce any apparent neurotoxicity. These studies provide the first in vivo evidence and proof-of-concept that SMART compounds are similarly efficacious to currently FDA approved antitubulin drugs for cancer treatment, but they can circumvent P-glycoprotein-mediated drug resistance.
tubulin; P-glycoprotein; pharmacokinetics; xenograft
Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Here we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospholipase D/autotaxin, agonists of the LPA5 GPCR, and blockers of lung metastasis of B16-F10 melanoma cells in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA5 compared to the R stereoisomer. In contrast, no stereoselective differences were found between the two isomers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These results extend the potential utility of these compounds as potential lead compounds warranting evaluation as cancer therapeutics.
lysophosphatidic acid; NPP2; autotaxin; GPR92; lysophospholipase D
A series of 2-aryl-4-benzoyl-imidazoles (ABI) was synthesized as a result of structural modifications based on the previous set of 2-aryl-imidazole-4-carboxylic amide (AICA) derivatives and 4-substituted methoxylbenzoyl-aryl-thiazoles (SMART). The average IC50 of the most active compound (5da) was 15.7 nM. ABI analogs have substantially improved aqueous solubility (48.9 μg/mL for 5ga vs. 0.909 μg/mL for SMART-1, 0.137 μg/mL for paclitaxel, and 1.04 μg/mL for Combretastatin A4). Mechanism of action studies indicate that the anticancer activity of ABI analogs is through inhibition of tubulin polymerization by interacting with the colchicine binding site. Unlike paclitaxel and colchicine, the ABI compounds were equally potent against multidrug resistant cancer cells and the sensitive parental melanoma cancer cells. In vivo results indicated that 5cb was more effective than DTIC in inhibiting melanoma xenograph tumor growth. Our results suggest that the novel ABI compounds may be developed to effectively treat drug-resistant tumors.
imidazole; ABI; SMART; melanoma; prostate cancer; antiproliferative activity; structure-activity relationship; tubulin polymerization inhibitor
Octadecenyl thiophosphate (OTP), a synthetic analog of the lysophospholipid growth factor lysophosphatidic acid (LPA), significantly reduces mortality following a lethal dose of LD80/30 radiation exposure in a mouse model of whole-body irradiation. To facilitate dose scaling between species, we developed a novel liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the preclinical pharmacokinetic characterization of OTP in monkeys. Sample extraction was carried out using a butanol based liquid-liquid extraction method. A partially deuterated OTP analogue was used as internal standard (IS). OTP and IS were separated by reversed-phase liquid chromatography on a C-8 column using 10 mM ammonium acetate and acetonitrile. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring was used to detect OTP and IS transitions of m/z 363.1→95.0 and 403.1→95.0. The method was applied to determine pharmacokinetic parameters in monkeys receiving a single oral OTP dose (3 mg/kg). OTP is readily absorbed with a relatively long half-life which supports further preclinical testing of OTP as a radioprotectant in monkeys.
Octadecenyl thiophosphate (OTP); lysophosphatidic acid (LPA); liquid chromatography/tandem mass spectrometry (LC-MS/MS); pharmacokinetics
Androgen receptor (AR) function is critical for the development of male reproductive organs, muscle, bone and other tissues. Functionally impaired AR results in androgen insensitivity syndrome (AIS). The interaction between AR and microRNA (miR) signaling pathways was examined to understand the role of miRs in AR function. Reduction of androgen levels in Sprague-Dawley rats by castration inhibited the expression of a large set of miRs in prostate and muscle, which was reversed by treatment of castrated rats with 3 mg/day dihydrotestosterone (DHT) or selective androgen receptor modulators. Knockout of the miR processing enzyme, DICER, in LNCaP prostate cancer cells or tissue specifically in mice inhibited AR function leading to AIS. Since the only function of miRs is to bind to 3′ UTR and inhibit translation of target genes, androgens might induce miRs to inhibit repressors of AR function. In concordance, knock-down of DICER in LNCaP cells and in tissues in mice induced the expression of corepressors, NCoR and SMRT. These studies demonstrate a feedback loop between miRs, corepressors and AR and the imperative role of miRs in AR function in non-cancerous androgen-responsive tissues.
We recently discovered a new class of thiazole analogs that are highly potent against melanoma cells. To expand the structure-activity relationship study and to explore potential new molecular scaffolds, we performed extensive ligand-based virtual screening against a compound library containing 342,910 small molecules. Two different approaches of virtual screening were carried out using the structure of our lead molecule: 1) connectivity-based search using Scitegic Pipeline Pilot from Accelerys and 2) molecular shape similarity search using Schrodinger software. Using a testing compound library, both approaches can rank similar compounds very high and rank dissimilar compounds very low, thus validating our screening methods. Structures identified from these searches were analyzed, and selected compounds were tested in vitro to assess their activity against melanoma cancer cell lines. Several molecules showed good anticancer activity. While none of the identified compounds showed better activity than our lead compound, they provided important insight into structural modifications for our lead compound and also provided novel platforms on which we can optimize new classes of anticancer compounds. One of the newly synthesized analogs based on this virtual screening has improved potency and selectivity against melanoma.
Pregna-5,7-dienes and their hydroxylated derivatives can be formed in vivo when there is a deficiency in 7-dehydrocholesterol (7-DHC) Δ-reductase function, e.g., Smith-Lemli-Opitz syndrome (SLOS). Ultraviolet B (UVB) radiation induces photoconversion of 7-DHC to vitamin D3, lumisterol3 and tachysterol3. Two epimers (20R and 20S) of pregna-5,7-diene-3β,17α,20-triol (4R and 4S, respectively) were synthesized and their UVB photo-conversion products identified as corresponding 9,10-secosteroids with vitamin D-like and tachysterol-like structures, and 5,7-dienes with inverted configuration at C-9 and C-10 (lumisterol-like). The number and character of the products and the dynamics of the process were dependent on the UVB dose. At high UVB doses, the formation of multiple oxidized derivatives of the primary products, and the formation of 5,7,9(11)-triene, were observed. The production of vitamin D-like, tachysterol-like and luminosterol-like derivatives was also observed in human skin treated with 4R and 4S, and subjected to UV irradiation, as shown by RP-HPLC. Newly synthesized compounds inhibited melanoma growth in dose dependent manner, and some of them showed equal or higher potency than 1,25(OH)2D3. In summary, we have characterized for the first time the products of UV induced conversion of pregna-5,7-diene-3β,17α,20-triols and documented that the newly synthesized compounds have antiproliferative properties against melanoma cells.
secosteroids; UV radiation; lumisterol; tachysterol; SLOS; melanoma
A series of pyridinium lipids containing a heterocyclic ring and a nitrogen atom were synthesized to determine the structure-activity relationship for gene delivery. Pyrylium chloroaluminate was synthesized by monoacylation of mesityl oxide and converted into pyrylium hexafluorophosphate, which was used as the key intermediate for reaction with different primary amines, to yield hydroxyethylpyridinium hexafluorophosphate and aminoethylpyridinium hexafluorophosphate. Acylation of these pyridinium salts with different types of fatty acid chlorides afforded the final pyridinium lipids, which were mixed with a co-lipid, such as L-alpha-dioleoylphosphatidylethanolamine (DOPE) and cholesterol (Chol) to prepare cationic liposomes by sonication. These liposomes were mixed with plasmid DNA encoding enhanced green fluorescent protein (pCMS-EGFP) or luciferase (pDNA3-Luc) and transfected into Chinese Hamster Ovary (CHO) cells. Several factors including hydrophobic anchor chain length, anchor chain type, configuration of double bond, linker type, co-lipid type, cationic lipid: co-lipid molar ratio, charge ratio (N/P), and cell type had significant influence on transfection efficiency and cytotoxicity. Pyridinium lipids with amide linker showed significantly higher transfection efficiency compared to their ester counterparts. Liposomes prepared at 1:1 molar ratio of pyridinium lipid and co-lipid showed higher transfection efficiency when either DOPE or cholesterol was used as a co-lipid to prepare cationic liposomes for complex formation with plasmid DNA at 3:1(+/−) charge ratio. Pyridinium liposomes based on hydrophobic anchor chain length of 16 showed higher transfection efficiency and lower cytotoxicity. The pyridinium lipid with trans-configuration of the double bond in fatty acid chain showed higher transfection efficiency than its counterpart with cis-configuration at the same fatty acid chain length. In the presence of serum, C16:0 and Lipofectamine significantly decreased their transfection efficiencies, which were completely lost at the serum concentration of 30% and higher, while C16:1 trans-isomer still had high transfection efficiency under these conditions. In conclusion, pyridinium lipids showed high transfection efficiency and have the potential to be used as transfection reagents in vitro and in vivo.
pyridinium lipid; cationic liposomes; gene delivery; plasmid; transfection
Calcitriol (3β,5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-1α,3β,25-triol) is a powerful oncostatic form of vitamin D3 that is of limited clinical utility due to hypercalcemic (toxic) effects. Since the removal of the side chain reduces or eliminates the calcemic activity of vitamin D3, secosteroidal compounds lacking or with a shortened side chain are good candidates for anti-cancer drugs. In addition, 5,7-steroidal dienes without a side chain can be generated in vivo under pathological conditions. A series of androsta- and pregna-5,7-dienes was efficiently synthesized from their respective 3-acetylated 5-en precursors by bromination-dehydrobromination and deacetylation reactions. Ultraviolet B (UVB) irradiation was used to generate corresponding 9,10-secosteroids with vitamin D-like structures. Additional products with tachysterol-like (T-like) structures or 5,7-dienes with inverted configuration at C-9 and C-10 (lumisterol, L-like) were also detected. Different doses of UVB resulted in formation of various products. At low doses, previtamin D-, T- or L-like compounds were formed as the main products, while higher doses induced further isomerization, with formation of potentially oxidized derivatives. In summary, we describe dynamic UVB induced conversion of androsta- and pregna-5,7-dienes into vitamin D-like compounds and their rearranged analogues; additionally novel T-like and L-like structures were also produced and characterized. Further biological evaluation of newly synthesized compounds should help to select the best candidate(s) for potential treatment of hyperproliferative diseases including cancer.
We have previously reported substituted 2-aryl-thiazolidine-4-carboxylic acid amides as potent and selective antiproliferative agents for melanoma. To understand the importance of the thiazolidine ring and to reduce potential complications associated with the two chiral centers, we designed and synthesized sets of new analogs by modifying this ring. These new analogs were tested in two melanoma cell lines and fibroblast cells (negative controls). Compared with the older analogs containing the thiazolidine ring, these new analogs have lower potency in general, but some of these analogs still have very good selectivity. These structure activity studies indicated that the thiazolidine ring is very critical for the activity for these series of compounds.