At a dose of 1 μM, TPPB (compound 5e) dramatically increased sAPPα secretion using a cell line obtained from Alzheimer's disease patients [1]. In PC12 cells, TPPB causes neurotoxicity due to anti-Aβ25-35. At a dosage of 1 μM, TPPB can counteract the cell damage caused by Aβ25-35. Treatment with Aβ25-35 inhibits the phosphorylation of Akt, PKC, MARCKS, and MAPK; TPPB increases this phosphorylation. Aβ25-35-induced caspase-3 activation is inhibited by TPPB [2].

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By use of a cell line derived from an AD patient, significant enhancement of sAPPα secretion was achieved at 1 μM concentration for most of the compounds studied and at 0.1 μM for TPPB (compound 5e) and compound 5f. At 1 μM the enhancement of sAPPα secretion for compounds 5c−h is higher than that observed for the control compound 8-(1-decynyl)benzolactam (BL). Of interest is the absence of activity found for the highly lipophilic ligand 5i, which has a Kiof 11 nM. On the other hand, its saturated counterpart 5j, which possesses a comparable Ki and ClogP, retains activity in the secretase assay. In the hyperplasia studies, 5f showed a modest response at 100 μg and 5e at 300 μg, suggesting that 5f was approximately 30-fold less potent than the PKC activator mezerein and 100-fold less potent than TPA. 5e was approximately 3-fold less active than 5f. On the basis of the effect of unsaturation for other potent PKC ligands, we would predict that 5e would retain biological activity in most assays but would show a marked loss of tumor-promoting activity. Compound 5e thus becomes a viable candidate compound in the search for Alzheimer's therapeutics capable of modulating amyloid processing.[1]

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In previous study, researchers reported that protein kinase C (PKC) activator TPPB could regulate APP processing by increasing α-secretase activity. In this study they further investigated the potential neuroprotective effect of TPPB against Aβ(25-35)-induced neurotoxicity in PC12 cells. The results indicated that TPPB at concentration of 1 μM could antagonize Aβ(25-35) induced cell damage as evidenced by MTT assays, LDH release and by morphological changes. Furthermore, the neuroprotection in cell viability can be blocked by inhibitors of PKC, Akt and MAPK. The experiment also indicated that TPPB could increase the phosphorylation of Akt, PKC, MARCKS and MAPK, which were inhibited by Aβ(25-35) treatment. Finally, TPPB inhibited the activation of caspase-3 induced by Aβ(25-35). Taken together, the experiment here implies that TPPB has a role against Aβ(25-35)-induced neurotoxicity in PC12 cells and may suggest its therapeutic potential in AD. [2]

Hyperplasia Assays.[1]
TPPB (compound 5e) and 5f were evaluated for induction of hyperplasia after topical application to the shaved backs of outbred Sencar mice. The extent of hyperplasia was estimated in terms of the number of cell layers in the epidermis (Table 2). The potencies of 5e and 5f were compared with those of TPA and of mezerein. Detectable hyperplasia was observed after a single application of 1 μg of TPA or after a single application of 3 μg of mezerein. 5f showed a modest response at 100 μg and 5e at 300 μg, suggesting that 5f was approximately 30-fold less potent than mezerein and 100-fold less potent than TPA. 5e was approximately 3-fold less active than 5f. Similar relationships were observed after four applications, although the extent of hyperplasia was somewhat more marked.

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After topical administration to the shaved backs of outbred Sencar mice, TPPB was assessed to induce hyperplasia and showed a moderate response at 300 μg [1].

sAPPα Determinations. [1]
The concentration of secreted sAPPα was measured using conventional immunoblotting techniques, following with minor modifications the protocol described elsewhere. Precipitated protein extracts from each dish/treatment were loaded into freshly prepared 8% acrylamide Tris-HCl minigels and separated by SDS−PAGE. The volume of sample loaded was corrected for total cell protein per dish. Proteins were then electrophoretically transferred to PVDF membranes. Membranes were saturated with 5% nonfat dry milk to block nonspecific binding. Blocked membranes were incubated overnight at 4 °C with the commercially available antibody 6E10 (1:500), which recognizes sAPPα in the conditioned medium. After being washed, the membranes were incubated at room temperature with horseradish peroxidase conjugated antimouse IgG secondary antibody. The signal was then detected using enhanced chemiluminescence followed by exposure of Hyperfilm ECL. The band intensities were quantified by densitometry using a BioRad GS-800 calibrated scanning densitometer and Multianalyst software.

24 h after plating 2×104 pheochromocytoma PC12 cells in each well of a 96 well plate, cells are incubated with TPPB at a series of concentration (0.1, 0.5, 1, 5, 10, 20 μM). Twelve to 24 h later, the original media is replaced with media containing MTT at a final concentration of 0.5 g/L for 4 h. Cell viability is evaluated with MTT assays[2].

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When cell viability, LDH and caspase-3 were tested, cells were pretreated with 1 μM TPPB (dissolved in DMSO) or vehicle for 1 h then with 20 μM Aβ for 24 h. When protein phosphorylation were detected, cells were pretreated with 1 μM TPPB for 1 h and then with 20 μM Aβ for 2 h. When various kinase inhibitors were used, they were added 30 min prior to TPPB treatment and then exposure to Aβ insult. For phospho-kinases assays, the original culture media was replaced with DMEM containing 0.5 % FBS 24 h before drug treatment. [2]

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MTT Assays [2]
Cell viability was evaluated with MTT assays. Briefly, 24 h after plating 2 × 104 cells in each well of a 96 well plate, cells were incubated with drugs at a series of concentration. Twelve to 24 h later, the original media was replaced with media containing MTT at a final concentration of 0.5 g/L for 4 h. Then the media was discarded and DMSO was added for the colorimetric assay. Absorption was determined in a Tecan Sunrise Eliza-Reader at λ = 570/630 nm after automatic subtraction of background signals. The results were expressed as a percentage of control group cells.

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Lactate Dehydrogenase (LDH) Release Determination[2]
The LDH released from the damaged cells was assayed according the protocols provided by the manufacture. In brief, at the indicated time points after drug treatment, the cells were collected and centrifuge at 250 g for 10 min. Then 100 μL supernatant were carefully transferred into corresponding wells of an optically clear 96-well plate. After 100 μL Reaction Mixture were added to each well, the samples were incubated for 30 min at room temperature (Protected the plate from light). The absorbances at 500 nm were measured using a microtiter plate reader. The cytotoxicity percentage was calculated after automatic subtraction of background signals. The results were expressed as a percentage of control groups.

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Western Blotting Analysis[2]
Twenty micrograms of protein mixed with 5× loading buffer (0.313 M Tris–HCl (pH 6.8) at 25 °C, 10 % SDS, 0.05 % bromophenol blue, 50 % glycerol) and 20× reducing agent (2 M DTT) was boiled for 5 min and loaded onto a 7.5 % SDS–polyacrylamide electrophoresis gel. After electrophoresis, the protein was electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes). The membranes were saturated with 5 % non-fat milk and incubated with primary antibodies (1:10,000 for β-actin, 1:1,000 for phospho-PKC, 1:1,000 for phospho-MARCKS, 1:1,000 for phospho-Akt and Akt, 1:1,000 for phospho-MAPK and MAPK, 1:1,000 for caspase-3 in bovine serum album) at 4 °C overnight. After being washed for 30 min in tris-buffered saline (TBS) with gentle agitation, membranes were incubated with horseradish peroxidase-conjugated anti-mouse/rabbit IgG secondary antibodies in non-fat milk at room temperature for 1 h. Signals were developed using ECL Western Blotting Detection kit.

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Sandwich ELISA[2]
The activities of phospho-Akt, Akt and cleaved caspase-3 were measured with sandwich ELISA. Briefly, following drug treatment, cells were harvested and treated with lysis buffer. After sonication and centrifugation, the supernatants were transferred to new microtubes and stored at −70 °C for future use. Before detection, the samples were lyophilized at −160 °C for 20 h and resuspended in the sample buffer. Then the detection antibody, secondary antibody and the substrate were added as indicated in the manufacturer’s instructions. The signals were determined in a microtiter plate reader at 450 nm and the results were expressed as percentage of control groups.

Hyperplasia Studies.[1]
Mice were 7 weeks old at the beginning of the treatments and were in the resting phase of the hair cycle. Compounds were dissolved in 0.2 mL of acetone and either were applied once or else were applied twice weekly for a total of four applications. Two animals were treated at each dose of compound, and 72 h after the last application, the animals were euthanized. Two portions of treated skin were removed from each animal, fixed in neutral buffered formalin, and stained with hematoxylin and eosin for histological analysis (staining of sections was performed by American Histolabs, Gaithersburg, MD). TPA (12-O-tetradecanoylphorbol 13-acetate) and mezerein were used.

[1]. New amide-bearing benzolactam-based protein kinase C modulators induce enhanced secretion of the amyloid precursor protein metabolite sAPPalpha. J Med Chem. 2003 Jan 30;46(3):364-73.

[2]. Neuroprotective effects of new protein kinase C activator TPPB against Aβ25-35 induced neurotoxicity in PC12 cells. Neurochem Res. 2012 Oct;37(10):2213-21.

Protein kinase C (PKC) is known to participate in the processing of the amyloid precursor protein (APP). Abnormal processing of APP through the action of the beta- and gamma-secretases leads to the production of the 39-43 amino acid Abeta fragment, which is neurotoxic and which is believed to play an important role in the etiology of Alzheimer's disease. PKC activation enhances alpha-secretase activity, which results in a decrease of the amyloidogenic products of beta-secretase. In this article, we describe the synthesis of 10 new benzolactam V8 based PKC activators having side chains of varied saturation and lipophilicity linked to the aromatic ring through an amide group. The K(i) values measured for the inhibition of phorbol ester binding to PKCalpha are in the nanomolar range and show some correlation with their lipophilicity. Compounds 5g and 5h show the best binding affinity among the 10 benzolactams that were synthesized. By use of a cell line derived from an AD patient, significant enhancement of sAPPalpha secretion was achieved at 1 microM concentration for most of the compounds studied and at 0.1 microM for compounds 5e (TPPB) and 5f. At 1 microM the enhancement of sAPPalpha secretion for compounds 5c-h is higher than that observed for the control compound 8-(1-decynyl)benzolactam (BL). Of interest is the absence of activity found for the highly lipophilic ligand 5i, which has a K(i) of 11 nM. On the other hand, its saturated counterpart 5j, which possesses a comparable K(i) and ClogP, retains activity in the secretase assay. In the hyperplasia studies, 5f showed a modest response at 100 microg and 5e at 300 microg, suggesting that 5f was approximately 30-fold less potent than the PKC activator mezerein and 100-fold less potent than TPA. 5e was approximately 3-fold less active than 5f. On the basis of the effect of unsaturation for other potent PKC ligands, we would predict that 5e would retain biological activity in most assays but would show a marked loss of tumor-promoting activity. Compound 5e thus becomes a viable candidate compound in the search for Alzheimer's therapeutics capable of modulating amyloid processing.[1]
In light of the present findings, we believe that compounds such as 5e (TPPB) that are capable of enhancing sAPPα secretion are worth exploring further as possible therapeutics in the treatment of Alzheimer's disease. Such compounds could be used alone or in combination with β-secretase inhibitors to decrease the formation of the neurotoxic β-amyloid peptide, thereby slowing the progression of the disease process [1].

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Alzheimer's disease (AD) is pathologically characterized by presence of senile plaques in the hippocampus, which are composed mainly of extracellular deposition of a polypeptide known as the beta amyloid, the Aβ. It has been demonstrated on numerous occasions that it was the deposition and aggregation of this Aβ peptide that cause neuronal dysfunction and even finally, the dementia. Lowering the deposition of Aβ or decreasing its neurotoxicity has long been one of the purposes of AD therapy.[2]
Collectively, the experiments here showed that the new PKC activator TPPB could protect PC12 cells from Aβ25–35 insult as indicated by cell viability detection and morphological changes. TPPB corrected Aβ25–35-attenuated Akt, PKC, MARCKS and MAPK phosphorylation and inhibit Aβ-induced caspase-3 activation through PKC-mediated signaling pathways. Activation of PKC with other PKC agonists such as PMA has been shown to increase sAPPα release and to reduce secretion of the Aβ peptide. But the carcinogenicity of PMA limits its clinical application. Other PKC agonists such as bryostatin 1 and methylazoxymethanol acetate all could increase sAPPα, but the precise mechanism how they activate α-secretase is unclear. TPPB could regulate APP processing by increasing α-secretase activity and decreasing Aβ secretion; here the data showed it could inhibit Aβ25–35-induced neurotoxicity. Considering the different roles of sAPPα and Aβ and the possible neuroprotective roles of above-mentioned kinases in AD pathophysiology, we speculate that TPPB may have therapeutic potential in AD, but further research is needed.[2]

C27H30N3O3F3

501.5406

501.223

C, 64.66; H, 6.03; F, 11.36; N, 8.38; O, 9.57

497259-23-1

9935767

Light yellow to yellow solid powder

1.2±0.1 g/cm3

728.8±60.0 °C at 760 mmHg

394.6±32.9 °C

0.0±2.5 mmHg at 25°C

1.572

3.09

3

7

6

36

806

2

CC(C)[C@H]1C(=O)N[C@@H](CC2=C(N1C)C=CC(=C2)NC(=O)/C=C/C=C/C3=CC=C(C=C3)C(F)(F)F)CO

WOLVEMPZUIFSII-IHHOKICGSA-N

InChI=1S/C27H30F3N3O3/c1-17(2)25-26(36)32-22(16-34)15-19-14-21(12-13-23(19)33(25)3)31-24(35)7-5-4-6-18-8-10-20(11-9-18)27(28,29)30/h4-14,17,22,25,34H,15-16H2,1-3H3,(H,31,35)(H,32,36)/b6-4+,7-5+/t22-,25-/m0/s1

(2E,4E)-N-[(2S,5S)-5-(hydroxymethyl)-1-methyl-3-oxo-2-propan-2-yl-2,4,5,6-tetrahydro-1,4-benzodiazocin-8-yl]-5-[4-(trifluoromethyl)phenyl]penta-2,4-dienamide

tppb; 497259-23-1; alpha-APP Modulator; (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam; CHEMBL331344; (2E,4E)-N-((2S,5S)-5-(hydroxymethyl)-2-isopropyl-1-methyl-3-oxo-1,2,3,4,5,6-hexahydrobenzo[e][1,4]diazocin-8-yl)-5-(4-(trifluoromethyl)phenyl)penta-2,4-dienamide; (2E,4E)-N-[(2S,5S)-5-(hydroxymethyl)-1-methyl-3-oxo-2-propan-2-yl-2,4,5,6-tetrahydro-1,4-benzodiazocin-8-yl]-5-[4-(trifluoromethyl)phenyl]penta-2,4-dienamide; PKC Activator V;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~249.23 mM)

Solubility in Formulation 1: ≥ 2.25 mg/mL (4.49 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 22.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.25 mg/mL (4.49 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 22.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)