Structural optimization of 10-methyl-aplog-1, a simplified analog of debromoaplysiatoxin,as an anticancer lead

Aplog-1 is a simplified analog of debromoaplysiatoxin (DAT) with potent tumor-promoting and proinflammatory activities. Aplog-1 and DAT exhibited anti-proliferative activities against several human cancer cell lines, whereas aplog-1 did not have tumor-promoting nor proinflammatory activities. We have recently found 10-methyl-aplog-1 (1) to have strong anti-proliferative activity compared with aplog-1. To further investigate the structural factors involved in the tumor-promoting, proinflammatory, and anti-proliferative activities, two dimethyl derivatives of aplog-1 (2, 3) were synthesized, where two methyl groups were installed at positions 4 and 10 or 10 and 12. 10,12-Dimethyl-aplog-1 (2) had stronger inhibitory effects on the growth of several human cancer cell lines than 1 and DAT, but exhibited no tumor-promoting and proinflammatory activities. In contrast, 4,10-dimethyl-aplog-1 (3) displayed weak tumor-promoting and proinflammatory activities along with anti-proliferative activity similar to that of 1 and DAT. Compound 2 would be the optimized seed for anticancer drugs among the simplified analogs of DAT.     

Synthesis and biological evaluation of the 12,12-dimethyl derivative of Aplog-1, an anti-proliferative analog of tumor-promoting aplysiatoxin

Aplog-1 is a unique analog of tumor-promoting aplysiatoxin that inhibits tumor-promotion by phorbol diesters and proliferation of tumor cells. While the structural features relevant to the biological activities of Aplog-1 remain to be identified, recent studies by us have suggested that local hydrophobicity around the spiroketal moiety of Aplog-1 is a crucial determinant of its anti-proliferative activity. This hypothesis led us to design 12,12-dimethyl-Aplog-1 (3), in which a hydrophobic geminal dimethyl group is installed proximal to the spiroketal moiety to improve biological potency. As expected, 3 was more effective than Aplog-1 in inhibiting cancer cell growth and binding to protein kinase Cδ, a putative receptor responsible for the biological response of Aplog-1. Moreover, an induction test on Epstein-Barr virus early antigen demonstrated 3 to be a better anti-tumor promoter than Aplog-1. These results indicate that 3 is a superior derivative of Aplog-1, and thus a more promising lead for anti-cancer drugs.     

Identification of protein kinase C isozymes involved in the anti-proliferative and pro-apoptotic activities of 10-Methyl-aplog-1, a simplified analog of debromoaplysiatoxin,in several cancer cell lines

10-Me-aplog-1 is a simplified analog of the tumor-promoting compound debromoaplysiatoxin (DAT) and a unique protein kinase C (PKC) activator with limited tumor-promoting and pro-inflammatory activities. 10-Me-aplog-1 inhibits the growth of several cancer cell lines, but the inhibitory mechanism involving PKC isozymes remains unclear. We quantified the amount of PKC isozymes in nine human cancer cell lines that differ in 10-Me-aplog-1 sensitivity. PKCα and δ were the predominant isozymes expressed in all cell lines, but there was no significant correlation between expression levels and anti-proliferative activity. Knocking down PKCα, and/or PKCδ in the three aplog-sensitive cell lines indicated their involvement in the anti-proliferative and pro-apoptotic activities of 10-Me-aplog-1. This finding suggests that PKCα and/or PKCδ activation could be effective for treating certain cancers. Since the mechanism underlying 10-Me-aplog-1's anti-proliferative activities resembles that of DAT, 10-Me-aplog-1 may be regarded as a special key derived from pleiotropic DAT as a bunch of keys.     

Artificial Analogs of Naturally Occurring Tumor Promoters as Biochemical Tools and Therapeutic Leads

Tumor promoters are non-carcinogenic chemicals that enhance tumor formation when administered repeatedly after a low dose of a carcinogen. Phorbol esters, teleocidins, and aplysiatoxins are typical examples of naturally occurring tumor promoters. All of them share the ability to bind and activate protein kinase C (PKC) despite the differences in their chemical structures. A variety of analogs with unique chemical and biological properties have been developed to analyze the molecular mechanism of tumor promotion through PKC activation. Moreover, coupled with the emerging significance of PKC in the pathological processes of Alzheimer's disease (AD) and acquired immune deficiency syndrome (AIDS) as well as cancer, several efforts have been made recently to generate analogs of tumor promoters with therapeutic potential. This review focuses on artificial analogs of phorbol esters, teleocidins, and aplysiatoxins, and discusses their potential as biochemical tools and therapeutic leads.     

Further Purification of Amylase Inhibitor Produced by Streptomyces sp.

The amylase inhibitor produced by Streptomyces sp. consisted of, at least, three kinds of inhibiting materials (inhibitor A, B and C). These inhibitors were separated by the Chromatographic techniques on Amberlite CG–120 and Dowex 1 × 2, and by paper chromatography etc.

Inhibitor A, B and C seemed to contain both carbohydrates and amino acids, but the specific activities (I. U./mg glucose) of the inhibitors A, B and C were different, that is, 18,800, 45,700 and 59,000, respectively. Also their ratios of amino acids to neutral sugars (expressed by moles of leucine per those of glucose) were approximately 0.09 (inhibitor A), 2.77 (inhibitor B) and 0.58 (inhibitor C).     

Role of the phenolic hydroxyl group in the biological activities of simplified analogue of aplysiatoxin with antiproliferative activity

The 18-deoxy derivative (3) of a simplified analogue (1) of aplysiatoxin with antiproliferative activity was synthesized to examine the role of the phenolic hydroxyl group at position 18 in the biological activities of 1. Compound 3 as well as 1 showed significant affinity for protein kinase Cδ (PKCδ), and the antiproliferative activity of 3 was slightly higher than that of 1. However, the anti-tumor-promoting activity of 3 was less than that of 1 in vitro, suggesting that the phenolic hydroxyl group of 1 is necessary for the anti-tumor-promoting activity but not for the binding of PKCδ and antiproliferative activity. Moreover, PKC isozyme selectivity of 3 was similar to that of 1, suggesting non-PKC receptors for these compounds to play some roles in the anti-tumor-promoting activity of 1.     

Synthesis and Tumor-promoting Activities of 12-Epi-phorbol-12,13-dibutyrate

12-Epi-phorbol-12,13-dibutyrate (1), the C12-epimer of the most frequently used phorbol ester probe, phorbol-12,13-dibutyrate (PDBu), has been synthesized from phorbol in 9 steps in order to investigate the structural requirements for tumor-promoting activity. Compound 1 showed about 100-fold weaker in vitro biological activities related to in vivo tumor promotion, Epstein-Barr virus early antigen (EBV-EA)-inducing ability, superoxide (O₂ ^-) generation-inducing ability, and binding to the protein kinase C (PKC) regulatory domain surrogate peptides. The results indicated that the β-stereochemistry at position 12 of the phorbol skeleton is important for optimal activity. Binding selectivity to each PKC C1 domain of 1 was almost equal to that of PDBu.     

Synthesis and evaluation of biological activities of vibsanin A analogs

Vibsanin A is an 11-membered vibsane diterpenoid and is reported to induce myeloid cell differentiation via activation of protein kinase C (PKC) without tumor-promoting activity. Therefore, vibsanin A is thought to be an attractive compound for acute myeloid leukemia (AML) therapy. In this study, we synthesized vibsanin A analogs and compared the activity of these compounds for PKC activation and myeloid cell differentiation. We found that the hydroxymethyl group in vibsanin A is an important substituent to induce differentiation of AML cells. Collectively, our results showed the biochemical features of vibsanin A and provided new insights into the development of new antileukemic drugs.     

5,7-Dodecadien-1-ol: Candidate for the Sex Pheromone of the Pine Moth

Ferredoxin-nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from Spinacia oleracea. The isolated enzyme was homogeneous by disc electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 86,000 by Ultrogel AcA 34 gel filtration. In the oxidized form, the enzyme had absorption maxima at 278, 388 (Soret band), 573 (α band) and 690 nm, indicating that siroheme is directly involved in the catalysis of nitrite reduction. This absorption spectrum was modified by sulfite, hydroxylamine and cyanide. The enzyme exhibited electron paramagnetic resonance signals with g values of 6.9 and 5.2, which are characteristic of a high spin Fe³⁺ -siroheme in the molecule. These signals disappeared upon the addition of dithionite or nitrite. This isolated enzyme also contained four moles of labile sulfide and 7 g-atoms of iron per 86,000 g of protein.     

Synthesis and evaluation of the anti-proliferative and NF-κB activities of a library of simplified tylophorine analogs

Tylophorine and many related phenanthropiperidine alkaloids are extraordinarily potent anti-proliferative agents. Despite their impressive anti-cancer activity, clinical development of these alkaloids has been hampered by their poor solubility and neurological side effects. Although it has been suggested that developing polar phenanthropiperidines will mitigate these undesired properties, the lack of practical methods for the synthesis of such analogues has limited this effort. Here, we present a concise synthetic approach to N-substituted phenanthropiperidines, which enabled a systematic investigation of structure-activity relationships at an underexplored region of the tylophorine scaffold. This work suggests that ring E of tylophorine is essential for the anti-proliferative activity of the 6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline core scaffold.     

Synthesis and biological activities of a phosphorodithioate analog of 2',5'-oligoadenylate.

To enhance the resistance of 2-5A (pppA2'p5'A2'p5'A) to degradation by exo- and endonucleases, a phosphorodithioate analog was synthesized using a solid-phase phosphite triester approach with N6-benzoyl-5'-O-dimethoxytrityl-3'-O-t-butyldimethylsilyladenosine 2'-[S-(beta-thiobenzoylethyl)-pyrrolidinophosphorothioamidit e]. 5'-Monophosphorylation was accomplished with 2-[2-(4,4'-dimethoxytrityloxy)-ethylsulfonyl]ethyl-(2-cyanoe thyl)-(N,N- diisopropyl)-phosphoramidite. The resulting product, p5'A2'(s2p)- 5'A2'(s2p)5'A, was approximately 10-fold less effective as an activator of purified human recombinant 2-5A-dependent RNase than was 2-5A itself. This loss of activation ability was related directly to the loss of binding ability of the phosphorodiothioate analog. As predicted, p5'A2'(s2p)5'A2' (s2p)5'A was stable to snake venom phosphodiesterase and the nucleolytic activities of both human lymphoblastoid CEM cell extracts and human serum, under conditions that led to facile degradation of parent 2-5A. This nuclease stability permitted the observation of the CEM cell extracts and human serum phosphatase activity which led to 5'-dephosphorylation of p5'A2'(s2p)5'A2'(s2p)5'A.     

Transformation by the v-fms oncogene product: an analog of the CSF-1 receptor

The product of the c-fms proto-oncogene is related to, and possibly identical with, the receptor for the macrophage colony-stimulating factor, M-CSF (CSF-1). Unlike the product of the v-erbB oncogene, which is a truncated version of the EGF receptor, the glycoprotein encoded by the v-fms oncogene retains an intact extracellular ligand-binding domain so that cells transformed by v-fms express CSF-1 receptors at their surface. Although fibroblasts susceptible to transformation by v-fms generally produce CSF-1, v-fms-mediated transformation does not depend on an exogenous source of the growth factor, and neutralizing antibodies to CSF-1 do not affect the transformed phenotype. An alteration of the v-fms gene product at its extreme carboxyl-terminus represents the major structural difference between it and the c-fms-coded glycoprotein and may affect the tyrosine kinase activity of the v-fms-coded receptor. Consistent with this interpretation, tyrosine phosphorylation of the v-fms products in membranes was observed in the absence of CSF-1 and was not enhanced by addition of the murine growth factor. Cells transformed by v-fms have a constitutively elevated specific activity of a guanine nucleotide-dependent, phosphatidylinositol-4,5-diphosphate-specific phospholipase C. We speculate that the tyrosine kinase activity of the v-fms/c-fms gene products may be coupled to this phospholipase C, possibly through a G regulatory protein, thereby increasing phosphatidylinositol turnover and generating the intracellular second messengers diacylglycerol and inositol triphosphate.     

Design, synthesis and cytotoxic studies on the simplified oxy analog of eleutherobin

A straight forward entry into nine membered B ring of eleutherobin as oxy analog and its cyctotoxic properties on HBL cell lines is described. Molecular modeling studies were carried out to ascertain the binding of the model compound to the active site of beta-tubulin.     

Changes of PKA and PDK1 in the principal piece of boar spermatozoa treated with a cell-permeable cAMP analog to induce flagellar hyperactivation

A cAMP-induced protein tyrosine phosphorylation and flagellar hyperactivation are controlled via complicated signaling cascades in mammalian spermatozoa. For instance, these events seem to be regulated positively by the PKA-mediated signaling and negatively by the PI3K/PDK1-mediated signaling. In this article, we have shown molecular changes of PKA and PDK1 in cAMP analog (cBiMPS)-treated boar spermatozoa in order to disclose possible roles of these kinases in protein tyrosine phosphorylation and hyperactivation. Ejaculated spermatozoa were incubated with cBiMPS, and then they were used for biochemical analyses of sperm kinases by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The first 30-min incubation with cBiMPS highly activated PKA of the principal piece to the accompaniment of autophosphorylation on Thr-197 of catalytic subunits. However, protein tyrosine phosphorylation and hyperactivation were fully induced in the sperm samples after the 180-min incubation. A potentially active form of PDK1 (54/55-kDa phospho-PDK1) was detected in the principal piece of the spermatozoa during the 90-min incubation. Another potentially active form (59-kDa phospho-PDK1) gradually increased during the same incubation period. However, the PDK1 suddenly became inactive by the dephosphorylation after the 180-min incubation, namely coincidently with full induction of protein tyrosine phosphorylation and hyperactivation. Additionally, existence of PI3K-dependently suppressing mechanisms for protein tyrosine phosphorylation was confirmed in the principal piece by pharmacological experiments with LY294002 and biochemical analyses with anti-PI3K p85 antibodies. These findings suggest that dephosphorylation of PDK1 may be a molecular switch for enhancement of protein tyrosine phosphorylation and flagellar hyperactivation in boar spermatozoa.     

Effects of the methoxy group in the side chain of debromoaplysiatoxin on its tumor-promoting and anti-proliferative activities

Debromoaplysiatoxin (DAT) is a tumor promoter isolated from sea hare and exhibits anti-proliferative activity against several cancer cell lines. To clarify key residues that are responsible for its tumor-promoting activity, we focused on the chiral methoxy group in the side chain, whose role had not yet been discussed or examined before. Demethoxy-DAT (8) was derived from DAT and we evaluated its tumor-promoting activity, anti-proliferative activity, and ability to bind to protein kinase C (PKC) isozymes. Compound 8 showed somewhat weaker tumor-promoting activity than that of DAT both in vitro and in vivo, but showed higher anti-proliferative activity against several cancer cell lines. Although the affinity to novel PKC isozymes of 8 was comparable to that of DAT, the affinity to conventional PKC isozymes decreased slightly. These results suggest that the methoxy group of DAT is one of the key residues critical for tumor-promoting activity but not for anti-proliferative activity. Since the methoxy group has little influence on the molecular hydrophobicity, this is the first report showing that structural factors other than hydrophobicity in the side chain of DAT affected its biological activities.     

Synthesis of a potent enkephalin analog, Tyr-D-Thr-Gly-Phe-delta 3 Pro-NH2, and some of its biological activities

The enkephalin analog, H-Tyr-D-Thr-Gly-Phe-delta 3 Pro-NH2 X HCl (VI) ([D-Thr2, delta 3 Pro5]-enkephalinamide), has been synthesized by conventional methods in solution and purified to homogeneity by reversed phase HPLC. The analog is a potent analgesic agent. For evaluation of some of its biological activity a related compound [D-Thr2, Thz5]-enkephalinamide (XI) was also synthesized in solution. Anti-diarrhea activity was evaluated in mice by the intravenous route for anti-DL-5-hydroxytryptophan (5-HTP) induced diarrhea activity. Analgesic activity was assayed by the method of Nilsen in mice using the intravenous route, and by a modified tail flick test in rats and the acetic acid writhing test in mice following subcutaneous administration. Within the constraints of the assays the two analogs are approximately equipotent. Both are less active than [D-Ala2, MePhe4, Met(0)ol]-enkephalinol (XII). Earlier receptor binding studies of compound XI indicated enhanced affinity for the mu receptor and little for the delta receptor. By comparison this may also be the case for compound VI.     

Synthesis,and anti-proliferative,Pim-1 kinase inhibitors and molecular docking of thiophenes derived from estrone

Heterocyclization of steroids were reported to give biologically active products where ring D modification occured. Estrone (1) was used as a template to develop new heterocyclic compounds. Ring D modification of 1 through its reaction with cyanoacetylhydrazine and elemental sulfur gave the thiophene derivative 3. The latter compound reacted with acetophenone derivatives 4a-c to give the hydrazide-hydrazone derivatives 5a-c, respectively. In addition, compound 3 formed thiazole derivatives through its first reaction with phenylisothiocyanate to give the thiourea derivative 9 followed by the reaction of the later with α-halocarbonyl compounds. In the present work a series of novel estrone derivatives were designed, synthesized and evaluated for their in vitro biological activities against c-Met kinase, and six typical cancer cell lines (A549, H460, HT-29, MKN-45, U87MG and SMMC-7721). The most promising compounds 5b, 5c, 11a, 13c, 15b, 15c, 15d, 17a and 17b were further investigated against the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR. Compounds 5b, 15d, 17a and 17b were selected to examine their Pim-1 kinase inhibition activity where compounds 15d and 17b showed high activities. Molecular docking of some of the most potent compounds was demonstrated.     

Synthetic hispidin, a PKC inhibitor, is more cytotoxic toward cancer cells than normal cells in vitro

The trypanocidal activity of naturally occurring 6-(3,4-dihydroxystyryl)-4-hydroxy-2-pyrone (hispidin) prompted us to examine its cytotoxic activity toward normal and cancerous cells in culture. Hispidin synthesized in our laboratory to a high degree of purity (checked by 1H and 13C NMR spectroscopy) was shown to be cytotoxic (between 10-3 mol/L and 10-7 mol/L) toward normal human MRC-5 fibroblasts, human cancerous keratinocytes (SCL-1 cell line), and human cancerous pancreatic duct cells (Capan-1 cell line). Interestingly, addition of hispidin in three successive doses (between 10-5 mol/L and 10-7 mol/L) led to a 100-fold increase in activity with an enhanced activity on cancer cells compared to normal cells (50%). Synthetic hispidin was found to inhibit isoform of protein kinase C (IC50 of 2 × 10-6 mol/L), but not E. coli and placental type XV alkaline phosphatases. The enhanced activity of hispidin toward the cancerous cell lines is discussed.     

Synthesis,biological evaluation and anti-proliferative mechanism of fluorine-containing proguanil derivatives

Proguanil, a member of biguanide family, has excellent anti-proliferative activities. Fluorine-containing compounds have been demonstrated to have super biological activities including enhanced binding interactions, metabolic stability, and reduced toxicity. In this study, based on the intermediate derivatization methods, we synthesized 13 new fluorine-containing proguanil derivatives, and found that 7a,7d and 8e had much lower IC₅₀ than proguanil in 5 human cancerous cell lines. The results of clonogenic and scratch wound healing assays revealed that the inhibitory effects of derivatives 7a,7d and 8e on proliferation and migration of human cancer cell lines were much better than proguanil as well. Mechanistic study based on representative derivative 7a indicated that this compound up-regulates AMPK signal pathway and downregulates mTOR/4EBP1/p70S6K. In conclusion, these new fluorine-containing derivatives show potential for the development of cancer chemotherapeutic drugs.