Cinnamic Acid Analogues Research Articles

Inert gas sparge leads to alternate reaction pathway.

The effect of sparging with an inert gas (argon) was evaluated during the investigation of the solution kinetics of an oxidation-prone amphiphilic drug containing a sulphide moiety. Samples stored with an air headspace in pH7 and 8 phosphate buffers at elevated temperatures and in the absence of light degraded to two main products, a sulphoxide and a cinnamic acid analogue. Initially, this appeared to be a sequential mechanism which could be blocked by removing oxygen. Instead, argon-sparge forced the direct degradation to the cinnamate, which was evidenced by the formation of a strong odour of sulphide. In addition, argon-sparged samples remained colourless, while those sparged with oxygen or stored with an air headspace turned yellow and had negligible odour. The half-lives for samples stored in pH 8 buffers at 93 degrees C at an initial drug concentration of 25 mg mL(-1) were 128 days (argon sparged), 86 days (air headspace), and 65 days (oxygen sparged). The results indicated that for the drug under study, sparging with an inert gas affected the mechanism as well as the rate of the reaction at elevated temperatures.

Stimulatory effect of cinnamic acid analogues on alpha1A-adrenoceptors in-vitro.

We have characterized the effects of cinnamic acid and its derivatives on alpha(1)-adrenoceptor subtypes. The cinnamic acid with a methoxyl group and/or a hydroxyl group showed the ability to stimulate radioactive glucose uptake into C(2)C(12) cells, a cell line that specifically expresses the alpha(1A)-adrenoceptor subtype of alpha(1)-adrenoceptors. However, cinnamic acid without chemical modification diminished the glucose uptake into C(2)C(12) cells. It was shown that methoxylation and/or hydroxylation of cinnamic acid had higher affinities for alpha(1A)-adrenoceptors investigated using [(3)H]prazosin binding experiments in C(2)C(12) cells. The effect of these derivatives on alpha(1A)-adrenoceptors was further characterized using the displacement of [(3)H]prazosin binding in rat prostate. We found that 3,5-dimethoxy-4- hydroxycinnamic acid, the cinnamic acid derivative with two methoxyl groups and hydroxylation at the fourth carbon on the benzene ring, had a higher affinity for the alpha(1A)-adrenoceptor subtype, showing a smaller IC50 value (the concentration for production of 50% inhibition) to displace [(3)H]prazosin binding in rat prostate. Affinity of these compounds for alpha(1B)-adrenoceptors was identified using [(3)H]prazosin-binding experiments in rat spleen. However, we found no marked differences in the IC50 values between these cinnamic acid analogues to displace the [(3)H]prazosin binding in rat spleen. In conclusion, our data indicated that methoxylation and/or hydroxylation of cinnamic acid might raise the affinity for alpha(1A)-adrenoceptors.

Concise bromodecarboxylation of cinnamic acids to β-bromostyrenes

A convenient salt-free approach to the halodecarboxylation of cinnamic acid analogs to β-bromostyrene was reported. This conversion was conducted at 0 °C in CH 2Cl 2 with only 10% of acetic acid. This protocol can be used for cinnamic acids with a variety of substituents, including β-methyl cinnamic acid ( 1d) in 60–81% yield, but not for hydroxyl- and p-methyl-cinnamic acids. Meanwhile, pyridine hydrobromide perbromide was used as bromide source to convert 1a– 2a in 59% yield.

A Phase II Study of Oral Panobinostat (LBH589) in Accelerated Phase (AP) or Blast Crisis (BC) Chronic Myeloid Leukemia (CML) Patients Resistant to ≥2 BCR-ABL Tyrosine Kinase Inhibitors

Panobinostat (LBH589), a novel cinnamic hydroxamic acid analogue with potent pan deacetylase inhibitor activity, acetylates HSP90 and promotes degradation of its client proteins, such as BCR-ABL. Panobinostat induces degradation of wild-type BCR-ABL, as well as BCR-ABL with the T315I and the E255K mutations in BaF3 cells. It also induces apoptosis in primary patients CML-BC cells. Panobinostat was investigated for the first time in patients (pts) in AP or BC stage of a Ph1+ CML in this Phase II trial. Panobinostat was dosed orally, 20 mg, once-a-day, thrice weekly. Concomitant hydroxyurea was permitted for up to 7 days within 14 first days in Cycle 1. Primary objective was to assess hematologic response rate, ie, complete hematologic response (CHR) with no evidence of leukemia (NEL) or return to chronic phase (RTC). Response confirmation was required after 4 weeks. As per protocol, to avoid futility for pts, efficacy cut-off required to move to Stage 2 enrollment was of 3 or more major cytogenetic responders (MCyR) out of the first 25 treated pts in Stage 1. A total of 27 pts, 13 female/14 male, with Ph+ CML who had failed ≥2 prior BCR-ABL tyrosine kinase inhibitors (TKIs), were enrolled into Stage 1 cohort of the study. Median age at entry was 55 years (29–76). Patients entered with a disease stage of AP or BC of 17/10, respectively. Time since initial CML diagnosis was ≥5 years in 59% of pts. The majority of pts (55%) had evolved into their current disease stage (AP or BC) within the year prior to entry. Best response under TKIs, at any disease stage, had been cytogenetic response (CyR) in 8 pts, complete hematologic response (CHR) in 9 pts, and finally, no CHR in 10 pts. Median treatment duration with panobinostat was 17 days (1–76). Discontinuation from study was due to disease progression in 19 pts (70%) or new leukemia therapy in 1 pt (3%), and it was recorded as due to AE in 5 pts (18%) or to abnormal lab values in 2 pts (7%). CNS new involvement by leukemia occurred in 4 pts. The most common Grade 3/4 AEs were thrombocytopenia in 12 pts (44%) and anemia in 9 pts (33%). Gastrointestinal (GI) AEs were Grade 1/2 for 13 of the 14 events, including diarrhea in 10 pts. 1 pt presented Grade 3 abdominal pain. Atrial fibrillation recorded as possibly related to study drug was the AE cause for discontinuation in 1 pt. Out of 684 records of post baseline ECGs evaluable to date, 1 single QTcF prolongation >480 ms was recorded (Grade 2). Notable lab abnormalities were Grade 3 hyperkalemia in 2 pts, Grade 3 hypokalemia in 2 pts, and Grade 3 WBC count increased in 2 pts. Hematologic RTC response was reported for 4 pts, (in Cycle 1 for 1 pt, Cycle 2 for 2 pts, and Cycle 3 for 1 pt). However, these responses were not confirmed. Consistently, no MCyR was observed. No major molecular response was observed from BCR-ABL transcript analysis performed at a central lab from patients blood samples obtained at baseline and post treatment. BCR-ABL mutation analysis by direct sequencing at a central lab showed that in addition to T315I, which was most frequently observed, a spectrum of other mutations were also seen at baseline and at disease progression. Some patients harbored more than one mutation in BCR-ABL at baseline and/or disease progression, which will be presented in details. Single-agent, oral panobinostat at a dose of 20 mg/day, thrice weekly did not show any sustained clinical responses in AP or BC CML patients enrolled. Furthermore, disease progression was very rapidly determined with median therapy duration being less than 3 weeks. This could possibly be related to a too short interval of temporary co-administration of hydroxyurea, as per study protocol. In patients with AP/BC CML resistant to 2 TKIs, oral, single-agent panobinostat at a dose of 20 mg/day, thrice weekly was safe but did not show meaningful clinical activity precluding study continuation. Promising clinical activity of panobinostat has since been observed in various other hematological malignancies, such as CTCL, as well as HL, AML, and MDS — mostly at higher doses. In these and other indications, clinical investigation of the oral and i.v. formulations of panobinostat at different doses and in combination with established standard treatment regimens are planned or ongoing.

The Novel Potent Pan-Deacetylase (DAC) Inhibitor, Panobinostat (LBH589), Markedly Enhances the Anti-Myeloma Effects of Chemotherapy In Vitro and In Vivo.

Deacetylase (DAC) inhibitors represent a new class of anti-cancer therapeutics that inhibit DAC enzymes and have been shown to have multiple effects in tumor cell lines including decreased oncoprotein expression (Bcr-Abl, HER-2), decreased angiogenesis, induction of apoptosis, induction of cell-cycle arrest, and decreased tumor cell motility and invasion. Panobinostat (LBH589), a novel cinnamic hydroxamic acid analogue with potent histone DAC inhibitor activity, has recently been shown to have the potential to treat a wide range of solid and hematological malignancies including multiple myeloma (MM). In this study, we first evaluated the in vitro anti-MM effects of panobinostat alone and in combination with doxorubicin or melphalan using the MM cell lines RPMI8226, U266 and MM1S. Cells treated with the combinations of panobinostat + doxorubicin and panobinostat + melphalan showed marked synergistic anti-MM effects as determined by measuring proliferation with the MTS assay compared to treatment with single agent and untreated cells. Next, we evaluated the anti-MM effects of panobinostat alone and in these combinations in vivo using one of our SCID-hu mouse models of human MM, LAGλ-1. Each SCID mouse was implanted with a 2.0 - 4.0 mm3 LAGλ-1 into the left superficial gluteal muscle. In our panobinostat single agent study, tumors were allowed to grow for 7 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into panobinostat treatment groups. Panobinostat was administered via intraperitoneal (i.p.) injection once daily five times per week at 5, 10 and 20 mg/kg. Control mice were given sterile normal saline as vehicle. Mice receiving panobinostat showed marked inhibition of tumor growth (10 mg/kg, P < 0.003; 20 mg/kg, P < 0.009) and reduction of paraprotein levels (10 mg/kg, P < 0.0025; 20 mg/kg, P < 0.015) compared to mice receiving vehicle. Next, we evaluated the combination of low-dose panobinostat (5 mg/kg) with low doses of either liposomal doxorubicin (1 mg/kg) or melphalan (3 mg/kg) i.p. in mice bearing LAGλ-1. Tumors were allowed to grow for 10 days at which time human IgG levels were detectable in the mouse serum, and mice were blindly assigned into treatment groups. Panobinostat was administered as above, and liposomal doxorubicin was injected once daily for three consecutive days weekly and melphalan once weekly. Mice treated with the combination of panobinostat + liposomal doxorubicin showed markedly smaller tumors and reduced hIgG levels compared to treatment with the DAC inhibitor alone, and treatment with liposomal doxorubicin as a single agent produced no anti-MM effects. Mice bearing LAGλ-1 treated with the combination of low-dose panobinostat + low-dose melphalan also showed markedly smaller tumors and decreased hIgG levels compared to treatment with panobinostat alone whereas mice receiving melphalan alone showed similar results to vehicle-treated animals. These promising results support the further clinical development of panobinostat and suggest that combining this DAC inhibitor with low-dose chemotherapy (liposomal doxorubicin or melphalan) may enhance the efficacy of this novel agent for the treatment of MM patients.

Molecular Characterization and Drug-Sensitivity of Pan Histone Deacetylase Inhibitor Resistant Human Acute Myeloid Leukemia Cells.

Hydroxamic acid analogue pan-histone deacetylase (HDAC) inhibitors (HA-HDIs), e.g., vorinostat, LAQ824 and LBH589, induce in vitro growth arrest, differentiation and apoptosis of human acute leukemia cells. Continuous and protracted use of HA-HDI, as currently used in the clinic against hematologic malignancies is likely to result in the emergence of HA-HDI resistance in leukemia cells. By continuous in vitro exposure of the AML HL-60 cells to the cinnamic acid analogue HA-HDI LAQ824, we have generated an in vitro and in vivo model of HA-HDI-resistant HL-60/LR cells, which are capable of growth in high concentrations (200 nM) of LAQ824. HL-60/LR versus the parental HL-60 cells have a shorter doubling time (12 versus 24 hours), increased % of cells in the S phase of the cell cycle (62.4 versus 40.0) and exhibit shorter interval to generation of leukemia and survival in NOD/SCID mice. As compared to HL-60, HL-60/LR cells have a resistance index of 100 for LAQ824, and are cross-resistant to other antileukemia agents exhibiting resistance index for LBH589: 50; trichostatin A: 15; vorinostat: 30; sodium butyrate: 10; etoposide: 5.0; Ara-C: 3.3 and TRAIL: 31.3. As compared to HL-60, HL-60/LR cells express higher levels of Bcl-xL and XIAP but lower levels of MCL-1. HL-60/LR versus HL-60 cells also express markedly reduced levels of Bim and Bak but higher levels of Bax. Although expressing higher levels of the death receptors (DR) 4 and 5 and lower levels of c-FLIP, HL-60/LR cells lack expression of caspase-8 and show barely detectable levels of FADD. Additionally, HL-60/LR versus HL-60 cells have markedly higher levels of AKT, c-RAF, and p-STAT5. Although expressing higher levels of HDAC1, HDAC2, and HDAC4, HL-60/LR cells lack detectable expression of HDAC6, with increased expression of hyper-acetylated hsp90 and α-tubulin- two of the substrates deacetylated by HDAC6. As compared to hsp90 in HL-60 cells, hyper-acetylated hsp90 in HL-60/LR cells exhibits less binding to ATP and p23. Utilizing a polyclonal antibody generated against acetylated hsp90α, confocal immunofluorescence microscopy showed higher and mostly cell surface expression of acetylated hsp90α in HL-60/LR versus HL-60 cells. As compared to HL-60, treatment of HL-60/LR cells with LAQ824 failed to induce p21 and hsp70, or increase the levels of hyper-acetylated hsp90 and α-tubulin. Notably, although cross-resistant to several anti-leukemia drugs, HL-60/LR cells are collaterally sensitive to the hsp90-inhibiting geldanamycin analogues 17-allylamino-demothoxy geldanamycin (17-AAG) and 17-DMAG with a four and five-fold increased sensitivity to 17-AAG and 17-DMAG, respectively. This was associated with a lack of both a 17-AAG mediated induction of hsp70 and a lesser decline in the levels of AKT and c-RAF in HL-60/LR versus HL-60 cells. Taken together, these findings elucidate several notable in vitro and in vivo biologic characteristics and drug-sensitivity profile of the first fully-characterized HA-HDI-resistant human AML cells. Our findings clearly demonstrate that in vitro resistance to HA-HDIs is associated with loss of HDAC6 expression, hyperacetylation of hsp90, aggressive leukemia phenotype, but cross-sensitivity to 17-AAG. These findings also suggest that hsp90 inhibitors should be tested for overriding de novo or acquired HA-HDI resistance in AML.

Direct profiling and imaging of peptides and proteins from mammalian cells and tissue sections by mass spectrometry.

Mass spectrometry can be used to map the distribution of targeted compounds in tissue, providing important molecular information in many areas of biological research. Matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS) is well suited for the analysis of tissue samples with a spatial resolution of about 30 microm for compounds in a mass range from 1000 to over 50 000 Da. Direct analysis of tissue sections requires spotting or coating of the tissue with a matrix compound typically sinapinic acid or other cinnamic acid analogs. A raster of this sample by the laser beam and subsequent mass analysis of the desorbed ions can record molecular intensities throughout the section. The overall process is illustrated by profiling and imaging of mouse epididymis sections where protein activity changes markedly throughout the section.

Direct profiling and imaging of peptides and proteins from mammalian cells and tissue sections by mass spectrometry

Mass spectrometry can be used to map the distribution of targeted compounds in tissue, providing important molecular information in many areas of biological research. Matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS) is well suited for the analysis of tissue samples with a spatial resolution of about 30 microm for compounds in a mass range from 1000 to over 50 000 Da. Direct analysis of tissue sections requires spotting or coating of the tissue with a matrix compound typically sinapinic acid or other cinnamic acid analogs. A raster of this sample by the laser beam and subsequent mass analysis of the desorbed ions can record molecular intensities throughout the section. The overall process is illustrated by profiling and imaging of mouse epididymis sections where protein activity changes markedly throughout the section.

Novel photoreactive cinnamic acid analogues to elucidate phenylalanine ammonia-lyase

4-[3-(Trifluoromethyl) diazirinyl] cinnamic acid derivatives were synthesized to elucidate properties of phenylalanine ammonia-lyase (PAL). 2-Methoxy and 2-biotinylated alkoxy compounds have inhibitory activity on the formation of phenylalanine from cinnamic acid. Specific photolabeling of the enzyme was detected using biotinylated derivatives without the use of radioisotopes. The results indicated that the 4-[3-(trifluoromethyl) diazirinyl] skeleton will be a suitable photoreactive compound to elucidate regulation of phenylpropanoid biosynthesis.

Anthocyanins from wild carrot suspension cultures acylated with supplied carboxylic acids

The anthocyanins accumulated by carrot cell cultures include some that are acylated. Addition of cinnamic and benzoic acid analogues to the culture medium resulted in the production of fourteen novel monoacylated anthocyanins. Four additional anthocyanins were prepared by partial acid hydrolysis, which selectively removed the xylose side chain from the branched trisaccharide part of the molecule. The carrot cells accommodate a wide range of carboxylic acids used to acylate anthocyanins and provide a system for the preparation of anthocyanins that have a variety of acyl groups attached. The compounds were characterized by 1H NMR spectroscopy and mass spectrometry, and by UV-vis absorption spectroscopy at both pH 2 and 6.

Synthesis of C-Alkylcalix[4]arenes. 4. Design, Synthesis, and Computational Studies of Novel Chiral Amido[4]resorcinarenes

In extending our studies involving BF3·Et2O-catalyzed reaction of cinnamic acid analogues, we have shown that amido derivatives also can afford [4]resorcinarene octamethyl ethers. Subsequently, chiral monomeric amides, derived from the mixed anhydride of cinnamic acid and l- or d-valine, upon treatment with BF3·Et2O, yielded for the first time chiral amido [4]resorcinarenes in enantiomerically pure forms. Four stereoisomers were isolated, and three of them were attributed the flattened-cone, chair, and 1,2-alternate conformations. The major product was assigned a novel chairlike structure, namely flattened partial cone 1. The flattened-cone stereoisomer, which was indicated by molecular modeling studies to be the most stable, became the major product under more drastic experimental conditions. Chromatographic studies on chiral phases revealed that the above tetramers could be used for the enantiodiscrimination of racemic molecules.

U-77,863: a novel cinnanamide isolated from Streptomyces griseoluteus that inhibits cancer invasion and metastasis.

Several cinnamoyl compounds have been shown to have antitumor activities, but not specifically anti-invasive or antimetastatic effects. U-77,863 (o-methyl cinnanamide) was originally isolated from a fermentation beer of Streptomyces griseoluteus and recently synthesized (Harper, DE and Welch DR. Journal of Antibiotics, in press). Based upon some differential activities of cinnanamides, in general, and U-77,863, specifically, we tested the hypothesis that U-77,863 could inhibit invasion and metastasis of human malignant melanoma cell lines C8161 and A375M. Pretreatment of melanoma cells in vitro with nontoxic doses of U-77,863 caused a dose-, and time-dependent, reversible reduction (IC50 = 12.5 micrograms/ml) of invasion through Matrigel-coated polycarbonate filters in the Membrane Invasion Culture System (MICS). Likewise, lung colonization was significantly (P < 0.05) inhibited when tumor cells were pretreated in vitro with U-77,863 prior to intravenous injection. Structure-activity analysis revealed that the acrylamide side-chain alone and cinnanamide were only slightly less potent than U-77,863, whereas cinnamic acid analogs did not inhibit tumor cell invasion at doses < or = 100 micrograms/ml. U-77,863 inhibits invasion and metastasis without decreasing growth rates or clonogenic potential. Adhesion to endothelial monolayers or extracellular matrices (Matrigel) is not affected by exposure to U-77,863. U-77,863 presumably inhibits metastasis by inhibiting tumor cell extravasation (invasion). U-77,863 is a lead compound for developing a novel class of anti-invasive/anti-metastatic drugs.