text:
Terpyridine derivatives
The invention pertains to terpyridine compounds having structure (I). These compounds form fluorescent lanthanide chelates with the appropriate metal ions. The fluorescent metal chelates are useful as probes in time-resolved fluorescence spectroscopy.
Latest Wallac Oy Patents:
- Device and a method for detecting a sample contained by a sample well
- Device and a method for managing a sample to be analyzed and a solid sample carrier and liquid sample carrier
- System and method for determining risk of diabetes based on biochemical marker analysis
- Method and a switch device for producing an electrical signal in response to mechanical force
- Method and apparatus for detecting elution of samples
The invention pertains to organic complexing agents (ligands or chelators) having a 2,2':6',2" terpyridine structure. Throughout this specification compounds having this structure will be called terpyridines, if not otherwise specified. The ligands of the invention form fluorescent lanthanide chelates with the appropriate metal ions.
The new chelates as well as the ligand as such may find application in those fields that are classical for chelates. The fluorescent metal chelates of our invention are useful as probes in time-resolved fluorescence spectroscopy. Contrary to many other known lanthanide chelators, the chelators of this invention very often give strongly fluorescent chelates with both Tb.sup.3+ and Eu.sup.3+. This might imply that they will be very important in multi labeling techniques.
The sensitivity of the analytical methods based on molecular fluorescence is limited by the background signal due to either Raman scattering or fluorescent impurities in the sample. Both of these interferences are generally short-lived phenomena, i.e. the radiative transition following the excitation of the molecule occurs within a microsecond time span. Thus any compound with a long-lived fluorescence can be effectively determined in the presence of short-lived background if a fluorometer with time resolution is at hand. In this fluorometer the sample is illuminated by an intermittent light source such that the long-lived fluorescence is measurable during the dark period subsequent to the decay of the short-lived background.
The chelating ability of terpyridines, bipyridines and substituted pyridines are well known EP-A-68,875 (p. 29), Chemical Abstracts 98 (1983) 226913k, 102 (1985) 71589j, 103 (1985) 71419z and 106 (1987) 94885, FR-A-2,570,703, Chem Berichte 118 (1985) 212-7, EP-A-195,413 and EP-A-203,047).
Time resolved fluorescence spectroscopy by the use of fluorescent lanthanide chelates has also been known for some time. The methods presented in DE-A-2,628,158 and U.S. Pat. No. 4,374,120 make use of beta-diketones as ligands in fluorescent lanthanide chelates used in fluoroimmunoassays. In DE-A-2,628,158 the light absorbing moiety of the fluorescent chelate is covalently conjugated to an antigen or antibody which during the test procedure is allowed to form an immune complex with its immunological counterpart. In this type of assays the test protocol is arranged so that the amount of the immune complex formed will constitute indication means for the substance that is to be detected (=analyte). The lanthanide chelate-labeled immune reactant used has an epitope in common with the analyte (analyte analogue) or is an antibody active component directed against the analyte or against other antigenic or haptenic material used for the formation of the immune complex. In many versions of this type of assays the chelate may be conjugated directly to a molecule employed as an antigen or hapten. There are several different ways of categorizing fluoroimmunoassays and other assays employing biospecific affinity reactions (reactants). If, before the fluorescence measurement, the chelate incorporated in the immune complex is separated from the portion of the chelate that is not incorporated, the methods are called heterogenous. If no such separation step is included they are called homogenous. These two variants put different demands on the lanthanide chelate used. The homogenous ones require that the chelate changes its fluorescent properties in a measurable manner as a consequence of the immune complex formation, while no such requirement is needed for the heterogenous versions. Immunoassays are just one example of methods that utilize biospecific affinity reactions. Analogously there are assay systems utilizing other types of biospecific affinity such as between DNA--complementary DNA, Protein A--Fc-part of IgG, lectin--carbohydrate structure etc. The principles and classification outlined above apply equally well to them.
The major short-coming of the fluorescent lanthanide chelates is that they rarely combine good aqueous stability with efficient fluorescence. In aqueous solvents their fluorescence is often quenched by water.
In EP-A-64,484 a version of a lanthanide chelate heterogenous fluoroimmunoassay is given that has no requirements for labels that are good fluorescers. Instead the interest is focused on the aqueous stability of the lanthanide chelate used. The ligand only serves to carry the lanthanide through the separation step, after which the lanthanide cat-ion is dissociated at a low pH and a highly fluorescent chelate is formed with an aromatic beta-diketone as ligand in a micellar phase. This method gives a very good sensitivity but suffers from a somewhat lengthy procedure.
It would be very advantageous to have highly fluorescent lanthanide probes with good aqueous stability. Such probes would allow homogenous assays and shorter assay procedures in heterogenous assays. They would also find use in other fluorescence methods, e.g. fluorescence microscopy.
Lanthanide chelates fluorescing in aqueous media have previously been suggested (EP-A-180,492 (macropolycyclic), EP-A-68,875 (phenols and aryl ethers and one specific terpyridine chelator), EP-A-195,413 (aryl pyridines) and EP-A-203,047 (ethynyl pyridines)). Although they to some degree can compensate for the short-comings set forth above only lanthanide chelates of the ethynyl pyridines (EP-A-203,047)have turned out to exhibit an efficient quantum yield and an aqueous stability for the commercialisation as directly fluorescing labels.
The compounds of the invention have the common structure given in formula I and comprise also ordinary analogues thereof, such as acid, ester, salt and chelate forms involving one or more of their chelating heteroatoms. ##STR1##
In formula I, n is an integer 0, 1, or 2, preferably 1, and specifies that the group X-Y is a substituent replacing a hydrogen or a group X.sub.1 -X.sub.5 in the parent terpyridine compound.
X.sub.1 -X.sub.5 are selected from
a) hydrogen
b) hydrocarbon group which may be straight, branched or cyclic, such as alkyl, alkenyl, alkynyl and an aromatic group (aryl), and containing additional structures not participating in the chelation such as aromatic ring systems, ether, thioether, ester, amido, amino (primary, secondary or tertiary), carboxy, halo, cyano etc., and other structures exhibiting heteroatoms,
c) cyano, halo and nitro, and
d) carboxylic acid (COOH), amido (CONH.sub.2), amino (NH.sub.2), hydroxy (OH), mercapto (SH) and substituted forms of these groups in which hydrogen has been replaced with a hydrocarbon group as defined in b) above, and the hydrogen of the amino and hydroxy group optionally also having the possibility of being replaced with an acyl group, RCO, in which R is an hydrocarbon group as defined in b) above.
Examples of alkyl are lower alkyl having less than 12, such as 1-5, carbon atoms.
Examples of different aromatic groups are phenyl, quinolyl, naphthyl, pyridyl, pyrimidyl etc. all of which may contain additional substituents. By selecting alkenyl, alkynyl or aromatic system directly bound or bound via a carbonyl, or via an uncharged nitrogen atom to anyone of the heteroaromatic rings in formula I, the conjugated electron system can be extended to more aromatic rings or carbon atoms. Preferably at most two aromatic ring structures are conjugated to the terpyridine structure.
The preferred X.sub.1 -X.sub.5 are hydrogen, hydroxyl, alkoxyl, amino, amido, alkyl, such as aminoalkyl, alkylthio, alkylamino, alkynyl, such as aminoethynyl, aryl, such as aminoaryl, aralkyl, aryloxyl, arylamino and arylethynyl, Fenyl(ene) is the preferred aryl moiety. From the synthetic point of view both X.sub.1 and X.sub.5 are preferably hydrogen.
Z.sub.1 and Z.sub.2 are chelating groups, that may be identical or different. Preferably Z.sub.1 and Z.sub.2 are --CH.sub.2 --Z' and --CH.sub.2 --Z", respectively, having the chelating ability confined to Z' and Z".
Each of Z.sub.1 and Z.sub.2 comprises two or more heteroatoms having a free pair of electrons and placed at a distance of two or three atoms from each other. Examples of efficient chelating heteroatoms are amino nitrogen atoms (primary, secondary and tertiary amines), negatively charged oxygen atoms e.g. in carboxylate anions (COO.sup.-), enolate anions (C.dbd.C--O.sup.-)phosphates or phosphonates. Another good chelating structure is the hydroxamate group (CONOH). In most cases the bridge between two neighbouring chelating heteroatoms contains 1, 2 or 3 aliphatic carbon atoms. Among particularly important Z' and Z" structures may be mentioned N-biscarboxymethyl amino and N-biscarboxyethyl amino groups, the analogous phosphate (--N(--CH.sub.2 --O--PO.sub.3.sup.2-).sub.2) and phosphonate (--N(--CH.sub.2 --PO.sub.3.sup.2-).sub.2) and 2,6dicarboxypiperidin-1-yl. Alternatively, Z.sub.1 and Z.sub.2 may form a bridge connecting the two outer pyridine rings giving a macrocyclic chelating compound. Preferably such a bridge is --CH.sub.2 N(CH.sub.2 COO.sup.-)CH.sub.2 --(Z'=Z"=NCH.sub. 2 COO.sup.-).
In some compounds of the invention the chelating heteroatoms (N and O) may exist as the corresponding protonated forms and for O also as ester forms, such as lower alkyl (C.sub.1 -C.sub.6) or benzyl esters.
From the spectrofluorometric point of view Z and Z' are preferably chelating to Eu.sup.3+, Tb.sup.3+, Dy.sup.3+ or Sm.sup.3+.
X-Y represents an inert organic group in which X is an inert and stable bridge and Y is (a) a functional group or (b) a residue of an organic compound (Y') that has properties retained in the compound of formula I (n=1 or 2) after it has been coupled covalently to the parent compound. The term "inert" above means that the group or bridge characterized by this adjective does not have any efficient chelating heteroatom closer than at a distance of four atoms from the heteroatoms participating in the chelation of a joint metal ion. In actual practice this means that the four atoms are normally represented by a four-carbon chain. By "stable" is meant that the bridge X does not deteriorate when the compounds of the invention are used, for instance the bridge does not easily undergo hydrolysis. In formula I, X-Y exist as a substituent replacing a hydrogen in a Z.sub.1 and/or Z.sub.2 group, preferably in a Z' and/or Z" group, or a hydrogen in one or two X.sub.1 -X.sub.5.
X may contain at least one structural element selected from among the following: --NR-- (secondary and tertiary amine), --CONR-- and --NRCO-- (substituted amide), --S--S-- (aliphatic disulfide), --S-- (aliphatic thioether), --O-- (ether), --COO-- and --OOC-- (ester), --N.dbd.N-- (diaza) and pure hydrocarbon chain which may be straight, branched or cyclic and contain from 1 to 12 carbon atoms. The carbon chain may be purely aliphatic or purely aromatic (including phenyl, naphthyl, quinolyl, pyridyl and bipyridyl), and other inert functional groups not participating in the chelation mentioned above. The symbol R in the substituted amide above represents preferably hydrogen but may be alkyl, for instance an alkyl having less than 5 carbon atoms.
Y may be selected from two main categories (A and B below):
A) Y may be the residue of an organic compound (Y') having certain properties which are substantially retained in the compound of formula I (n=1 or 2). The compound (Y') may be a biologically active molecule having the ability to participate in biospecific affinity reactions, such as between antigens (haptens) and the homologous antibody active components, nucleotides, aligonucleotides, complementary nucleic acids (RNA, DNA), lectins and carbohydrate structures, protein A and IgG etc. This type of biologically active molecules are often called targeting substances (targeting molecules). Usually they have been or can be easily derivatized to contain functional groups permitting conjugation to diversified types of compounds. The compound (Y') may also be a multifunctional organic compound being bound by one of its functional groups to the bridge X so as to leave at least one of its remaining functional groups free for further derivatization.
B) Y may be a functional group so selected that it can be made to react chemically with a functional group A of an organic compound (Y') so as to form a covalent linkage between Y' and a compound of formula I (n=1 or 2). The selection of Y deepens on A and vice versa, but it is believed that any artisan can make the proper selection of mutually reactive groups. Y and A may be selected from among electrophilic and nucleophilic groups. If they are a pair of electrophilic groups or a pair of nucleophilic groups, it is possible for instance to (a) employ oxidative coupling for forming the bond (e.g. --SH+HS----S--S--) or (b) convert one of the groups of the pair chemically to a group of the opposite type. An example of the latter case is the activation with bifunctional coupling reagents (also called activation reagents). If Y is nucleophilic and A electrophilic or vice versa these two groups can usually be reacted with each other without any preceding activation. Most nucleophilic groups comprise a heteroatom having an electron pair available for reaction with an electron deficient atom (electrophilic group).
Examples of suitable functional groups include isothiocyanato, bromoacetamido, iodoacetamido, succinamido, pyridyldithio, mercapto, cargoxyl and its active esters (e.g. N-hydroxysuccinimido or p-nitrophenyl), hydroxyl, aldehyde, amino, diazonium, tosyl, mesytylyl, trexyl, phosphodiester or phosphotriester. Other functional groups are known to those skilled in the art.
In a compound according to the invention it is imperative that all the groups mentioned can coexist. However, the reactive group Y does not necessarily have to coexist with a chelating group of the invention. For some purposes the chelating part of the molecule may be temporarily protected e.g. in the form of an ester so that the protected ligand will be coupled to the target molecule, and after deblocking may finally form the desired labelled product. The protective group is chosen in accordance with known principles (see for instance Protective Groups in Organic Synthesis; Greene, Tenn.; John Wiley & Sons Inc; USA (1981). Factors to be taken into account when the group is chosen are inter alia the stability of the compound with which Y is to be reacted, the reactivity of Y and the type of structure formed upon reaction of Y and the compound intended.
The examples of this specification given wellknown methods for introducing functionalized methylene groups next to a pyridine nucleus. These groups can subsequently be reacted with iminobisacetic esters to the formation of chelating groups Z.sub.1 and Z.sub.2. Phosphonate groups can be introduced according to J. Org. Chem. 31 (1966) 1603-- and phosphate groups according to Helv. Chim. Acta 70 (1987) 175. For the formation of chelates with different metal ions, see Nucl. Med. Biol. 13 (1986) 311-- and references cited therein. Prior to the first filing of this patent application no publication had presented any good method for the synthesis of iminobisacetic esters derivatives having a functionalized substituent at the methylene group of their acetyl moiety. A synthetic route has been developed at Wallac Oy, Finland and it will be exemplified in this specification in detail (examples 37-45. The so synthesized derivatives may be reacted with 6,6"-bishalomethyl terpyridines analogously to the method given in examples 34-35 below.
The preferred compounds of the invention have been summarized in formula II. ##STR2##
In formula II, X.sub.1 -X.sub.5, n, X and Y have the same meaning as stated before, except that at most two of X.sub.1 -X.sub.5 are groups other than hydrogen.
--------- means that X-Y is a substituent replacing a group X.sub.1 -X.sub.5 or H'. Ch is a chelating group selected from --COO.sup.-, --OPO.sub.3.sup.2-, and --PO.sub.3.sup.2-. This preferred aspect of the invention also comprises the acid, ester, salt and chelate forms as defined above.
In general terms one mode of our invention is to apply our new fluorescing chelates as probes in time-resolved fluorescent spectrometry. In one aspect the chelating light-absorbing moiety of the chelate is used as a fluorescence enhancer for methods in which substantially non-fluorescent Eu.sup.3+ --, Dy.sup.3+ --, Sm.sup.3+ -- or Tb.sup.3+ -- chelates are used. See for instance EP-A-64,484 (Wallac Oy) and U.S. Pat. No. 4,352,751 (Wieder). Another aspect is a time-resolved fluorometric method using a fluorescing lanthanide chelate as the probe for the determination of a target substance (structure). Generally these assay methods comprise two steps (i) marking specifically the target structure with a lanthanide (probe) chelate of this invention (or just the chelating ligand if the target is a lanthanide ion forming a fluorescing chelate with the ligant), and (ii) measuring by methods known per se the fluorescence related to said probe. Assay systems employing biospecific affinity reactions as given in the introductory part are one of the most important systems falling under this definition. Accordingly other steps may also be included and be performed before, between or after steps (i) and (ii). Compare also U.S. Pat. No. 4,058,732 (Wieder).
The invention is defined in the appended claims which are part of the description. The invention will now be illustrated with the synthesis of several compounds of the invention.
The structures and the synthetic routes employed in the experimental part are shown in reaction schemes 1-3. Schemes 1 and 2 illustrate terpyridine compounds having a 4'-respective 4- and 4"-substitutent(s). By the proper selection of R.sup.1 in the starting compounds substituents will be introduced permitting either direct coupling or coupling by the use of common bifunctional coupling reagents to a biological active molecule participating in biospecific affinity reactions. Examples of such groups are nucleophilic groups (primary amino, isothiocyanato etc). Scheme 3 illustrates the synthesis of iminobisacetic acid derivatives that can be used for the formation of the chelating groups Z.sub.1 and Z.sub.2 (of formula I). The derivatives contain a reactive group that cannot participate in the chelating but that can be used to link covalently a biological active molecule of the type as set forth above to the terpyridine structure. ##STR3##
1-(2-Pyridyl)-3-(3-nitrophenyl)-2-propenone, 3b
2-Acetylpyridine (1.82 g, 15.0 mmol) and 3-nitrobenzaldehyde (2.27 g, 15.0 mmol) were added to a solution of KOH (0.67 g, 12.0 mmol) in water (6 ml) and methanol (54 ml). The solution was stirred at room temperature for 2 hours, whereafter the precipitation of the product was complete. The precipitate was filtered and washed with methanol.
Yield: 93%
.sup.1 H NMR (CDCl.sub.3): 7.52-7.55 (1H, m); 7.62 (1H, t, J=8 Hz); 7.91 (1H, dt, J=8 Hz and 2 Hz); 7.94 (1H, d, J=16 Hz); 8.01 (1H, d, j=8 Hz); 8.21 (1H, d, J=8 Hz); 8.26 (1H, dd, J=8 Hz and 2 Hz); 8.44 (1H, d, J=16 Hz); 8.59 (1H, s); 8.78 (1H, d, J=5 Hz).
1-(2-Pyridyl)-3-(4-nitrophenyl)-2-propenone, 3a
This compound was synthesized using a method analogous to the synthesis described in example 1, 3b. The crude product was crystallized from ethanol.
Yield: 68%
.sup.1 H NMR (CDCl.sub.3): 7.52-7.56 (1H, m); 7.87 (2H, d, J=9 Hz); 7.91 (1H, dt, J=8 Hz and 2 Hz), 7.92 (1H, d, J=16 Hz); 8.21 (1H, d, J=8 Hz); 8.27 (2H, d, J=9 Hz); 8.43 (1H, d, J=16 Hz); 8.76 (1H, d, J=5 Hz)
1-(2-Pyridylcarbonylmethyl)pyridinium iodide, 5
2-Acetylpyridine (1.21 g, 10.0 mmol) and iodine (2.54 g, 10.0 mmol) were heated to reflux in pyridine (12 ml) for 1 hour. The solution was cooled, and the dark precipitate was filtered and washed with pyridine.
Yield: 72%
.sup.1 H NMR (DMSO): 6.51 (2H, s); 7.82-7.85 (1H, m); 8.08 (1H, d, J=7 Hz); 8.14 (1H, dt, J=8 and 2 Hz); 8.28 (2H, t, J=8 Hz); 8.73 (1H, t, J=8 Hz); 8.87 (1H, d, J=5 Hz); 9.00 (2H, d, J=6 Hz).
4'-(3-Nitrophenyl)-2,2';6',2"-terpyridine, 6c
1-(2-Pyridyl)-3-(3-nitrophenyl)-2-propenone (2.54 g, 10.0 mmol) and 1-(2-pyridylcarbonylmethyl)pyridinium iodide (3.26 g, 10.0 mmol) and ammonium acetate (4.82 g, 60.0 mmol) were heated to reflux in acetic acid (37 mol) for 1.5 hours whereafter the solution was cooled and the precipitate was filtered and washed with acetic acid. The crude product was crystallized from acetonitrile.
Yield: 56%
.sup.1 H NMR (DMSO): 7.54-7.58 (2H, m); 7.89 (1H, t, J=8 Hz); 8.06 (2H, dt, J=8 Hz and 2 Hz); 8.38-8.41 (1H, m); 8.41-8.44 (1H, m); 8.66 (1H, t, J=2 Hz); 8.70 (2H, d, J=8 Hz); 8.78-7.80 (4H, m).
4'-(4-Nitrophenyl)-2,2';6',2"-terpyridine, 6b
This compound was synthesized using a method analogous to the synthesis described in example 4, 6c. The crude product was purified with flash chromatography (silica, chloroform with methanol gradient).
Yield: 56%
.sup.1 H NMR (DMSO): 7.53-7.56 (2H, m); 8.05 (2H, dt, J=8 Hz and 2 Hz); 8.22 (2H, d, J=9 Hz); 8.40 (2H, d, J=9 Hz); 8.68 (2H, d, J=8 Hz); 8.76 (2H, s); 8.77 (2H, d, J=5 Hz).
2,2';6',2"-Terpyridine-N,N"-dioxide, 7a
2,2':6',2"-Terpyridine (0.94 g, 4.0 mmol) was dissolved in dichloromethane (80 ml) and m-chloroperbenzoic acid (2.60 g, 15.1 mmol) was added. After stirring overnight some dichloromethane was added and the solution was washed with 10% sodium carbonate. The dichloromethane phase was evaporated and purified with flash chromatography (silica, chloroform with methanol gradient).
Yield: 93%
.sup.1 H NMR (CDCl.sub.3): 7.31 (2H, dd, J=2 and 7 Hz); 7.40 (2H, dd, J=1 and 8 Hz); 7.98 (1H, t, J=8 Hz); 8.20 (2H, dd, J=2 and 8 Hz); 8.37 (2H, dd, J=1 and 7 Hz); 8.94 (2H, d, J=8 Hz).
UV (.lambda..sub.max in ethanol): 241, 279.
4'-(4-Nitrophenyl)-2,2';6',2"-terpyridine-N,N"-dioxide, 7b
This compound was synthesized using a method analogous to the synthesis described in Example 6, 7a. The crude product was purified by crystallization from a mixture of acetonitrile and methanol.
Yield: 51%
.sup.1 H NMR (DMSO): 7.52-7.58 (4H, m); 8.11 (2H, dd, J=7 Hz and 2 Hz); 8.26-8.28 (2 H, m); 8.41-8.46 (4H, m); 9.16 (2H, s).
4'-(3-Nitrophenyl)-2,2';6',2"-terpyridine-N,N"-dioxide, 7c
This compound was synthesized using a method analogous to the synthesis described in Example 6, 7a. The crude product was purified by crystallization from a mixture of ethanol and chloroform.
Yield: 78%
.sup.1 H NMR (DMSO): 7.52-7.58 (4H, m); 7.89 (1H, t, J=8 Hz); 8.25-8.28 (2H, m); 8.31 (1H, d, J=8 Hz); 8.38 (1H, dd, J=8 Hz and 1 Hz); 8.43-8.45 (2H, m); 8.61 (1H, t, J=2 Hz); 9.17 (2H, s).
6,6"-Dicyano-2,2';6',2"-terpyridine, 8a
2,2';6',2"-Terpyridine-N,N"-dioxide (7a) (0.96 g, 3.6 mmol) was suspended in dichloromethane (10 ml). N,N-dimethylcarbamyl chloride (0.95 g, 8.8 mmol) and trimethylsilyl cyanide (1.26 g, 12.7 mmol) were added. After stirring for two weeks at room temperature, 10% potassium carbonate was added until the solution was neutral. The phases were separated and the water phase was extracted with chloroform. The chloroform was evaporated and the residue was crystallized from a mixture of acetonitrile and tetrahydrofurane.
Yield: 65%
.sup.1 H NMR (CDCl.sub.3): 7.75 (2H, d, J=8 Hz); 8.01 (2H, t, J=8 Hz); 8.05 (1H, t, J=8 Hz); 8.58 (2H, d, J=8 Hz); 8.82 (2H, d, J=8 Hz)
UV (.lambda..sub.max in ethanol): 215, 250, 291.
4'-(4-Nitrophenyl)-6,6"-dicyano-2,2';6',2"-terpyridine, 8b
This compound was synthesized using a method analogous to the synthesis described in Example 9, 8a. The crude product was purified by crystallization from a mixture of acetonitrile and tetrahydrofurane.
Yield: 56%
.sup.1 H NMR (DMSO): 8.22 (2H, dd, J=8 Hz and 1 Hz); 8.30 (2H, d, J=9 Hz); 8.33 (2H, t, J=8 Hz); 8.43 (2H, d, J=9 Hz); 8.77 (2H, s); 8.01 (2H, dd, J=8 Hz and 1 Hz).
4'-(3-Nitrophenyl)-6.6"-dicyano-2,2';6',2"-terpyridine, 8c
This compound was synthesized using a method analogous to the synthesis described in Example 9, 8a. The crude product was purified by crystallization from a mixture of acetonitrile and tetrahydrofurane.
Yield: 71%
.sup.1 H NMR (DMSO): 7.90 (1H, t, J=8 Hz); 8.22 (2H, dd, J=8 Hz and 1 Hz); 8.33 (2H, t, J=8 Hz); 8.41-8.45 (1H, m); 8.47-8.72 (1H, m); 8.72 (1H, s); 8.77 (2H, s); 9.00 (2H, d, J=8 Hz).
6,6"-Bis(aminomethyl)-2,2';6',2"-terpyridine pentahydrochloride, 9a
Compound 8a (0.55 g, 1.9 mmol) was suspended in dry tetrahydrofurane (15 ml). The suspension was bubbled with nitrogen. Borane tetrahydrofurane complex (1M, 25 ml, 25.0 mmol) was slowly added. After stirring overnight, the extra borane was destroyed by adding methanol. The mixture was evaporated to dryness and the residue was dissolved in ethanol (30 ml) saturated with hydrochloric acid. After stirring for one hour the solution was cooled and the product was filtrated.
Yield: 54%
.sup.1 H NMR (D.sub.2 O): 4.68 (4H, s); 7.89 (2H, d, J=8 Hz); 8.31 (2H, t, J=8 Hz); 8.51 (2H, d, J=8 Hz); 8.82 (2H, d, J=8 Hz); 8.90 (1H, t, J=8 Hz)
UV (.lambda..sub.max in H.sub.2 O): 232, 293
4'-(4-Nitrophenyl)-6,6"-bis(aminomethyl)-2,2';6',2"-terpyridine pentahydrochloride, 9b
This compound was synthesized using a method analogous to the synthesis described in Example 12, 9a. The product was not purified and therefore no proper NMR data was taken. The crude product was used as such in the next step.
Yield: 56% of crude product
4'-(3-Nitrophenyl)-6,6"-bis(aminomethyl)-2,2';6',2"-terpyridine pentahydrochloride, 9c
This compound was synthesized using a method analogous to the synthesis described in Example 12, 9a. The crude product was used in the next step without further purifications.
Yield: 61% of crude product
6,6"-Bis[N,N-bis(t-butoxycarbonylmethyl)aminomethyl]-2,2';6',2"-terpyridine , 10a
Compound 9a (0.43 g, 0.9 mmol), acetonitrile (15 ml), dry sodium carbonate (2.00 g) and t-butyl ester of bromoacetic acid (1.44 g, 7.4 mmol) was refluxed for two hours and the salts were filtered. The organic phase was evaporated and purified with flash chromatography (silica, chloroform).
Yield: 38%
.sup.1 H NMR (CDCl.sub.3): 1.48 (36H, s); 3.56 (8H, s); 4.15 (4H, s); 7.66 (2H, d, J=8 Hz); 7.84 (2H, t, J=8 Hz); 7.90 (1H, t, J=8 Hz); 8.47 (2H, d, J=8 Hz); 8.50 (2H, d, J=8 Hz)
UV (.lambda..sub.max in ethanol): 235, 288.
4'-(4-Aminophenyl)-6,6"-bis[N,N-bis(ethoxycarbonylmethyl)aminomethyl]-2,2'; 6',2"-terpyridine, 10b
The corresponding nitrocompound was synthesized from 9b and an ethyl ester of bromoacetic acid using a method analogous to the synthesis described in 10a. The product was purified with flash chromatography (silica, chloroform with methanol gradient).
Yield: 20%
.sup.1 H NMR (CDCl.sub.3): 1.25 (12H, t, J=7 Hz); 4.18 (8H, q, J=7 Hz); 3.71 (8H, s); 4.23 (4H, s); 7.66 (2H, dd, J=8 Hz and 2 Hz); 7.90 (2H, t, J=8 Hz); 8.06 (2H, d, J=9 Hz); 8.42 (2H, d, J=9 Hz); 8.57 (2H, dd, J=8 Hz and 2 Hz); 8.76 (2H, s).
The aminocompound 10b was synthesized from the corresponding nitrocompound using a method analogous to the synthesis described in 10c.
Yield: 16%
4'-(3-Aminophenyl)-6,6"-bis[N,N-bis(ethoxycarbonylmethyl)aminomethyl]-2,2'; 6', 2"-terpyridine, 10c
The corresponding nitrocompound was synthesized from 9c and an ethyl ester of bromoacetic acid using a method analogous to the synthesis described in 10a. The product was purified with flash chromatography (silica, chloroform with methanol gradient).
Yield: 53%
.sup.1 H NMR (CDCl.sub.3): 1.24 (12H, t, J=8 Hz); 4.17 (8H, q, J=8 Hz); 3.69 (8H, s); 4.23 (4H, s); 7.67 (2H, dd, J=8 Hz and 2 Hz); 7.89 (2H, t, J=8 Hz); 7.85-8.30 (4 H, m); 8.57 (2H, dd, J=8 Hz and 2 Hz); 8.75 (2H, s).
The aminocompound 10c was synthesized from the corresponding nitrocompound (50 mg, 0.7 mmol) which was dissolved in methanol (2 ml). 10% Palladium on carbon (10 mg) was added followed by slow addition of sodium borohydride (4 mg, 0.1 mmol). After one hour the reaction mixture was filtered and the solution was evaporated. The residue was dissolved in 0.1M sodium hydroxide solution and the product was extracted with chloroform from the water phase. The organic phase was dried and evaporated. The product was purified with flash chromatography (CHCl.sub.3, MeOH; 10:0.5).
Yield: 79%
6,6"-Bis[N,N-bis(carboxymethyl)aminomethyl]-2,2';6',2"-terpyridine, 11a
Tetraester (10a) (260 mg, 0.4 mmol) was dissolved in trifluoroacetic acid (3 ml). After two hours the trifluoroacetic acid was evaporated, some diethyl ether was added and the product was filtered.
Yield: 87%
.sup.1 H NMR (DMSO): 3.99 (8H, s); 4.47 (4H, s); 7.65 (2H, d, J=8 Hz); 8.10 (2H, t, J=8 Hz); 8.11 (1H, t, J=8 Hz); 8.48 (2H, d, J=8 Hz); 8.61 (2H, d, J=8 Hz)
UV (.lambda..sub.max in water): 237, 288, 305.
4'-(4-Aminophenyl)-6,6"-bis[N,N-bis(carboxymethyl)aminomethyl]-2,2';6',2"-t erpyridine, 11b
This compound was synthesized using a method analogous to the synthesis described in Example 20, 11c.
Yield: 100%
.sup.1 H NMR (DMSO): 3.82 (8H, s); 4.31 (4H, s); 6.90 (2H, d, J=7 Hz); 7.67 (2H, d, J=8 Hz); 7.80 (2H, d, J=9 Hz); 8.08 (2H, t, J=8 Hz); 8.60 (2H, d, J=8 Hz); 8.67 (2H, s).
4'-(3-Aminophenyl)-6,6"-bis[N,N-bis(carboxymethyl)-aminomethyl]-2,2';6',2"- terpyridine, 11c
The corresponding tetraester, 10c (0.10 g, 0.17 mmol) was dissolved in 5 ml 0.5M KOH-EtOH solution. After 2 hours stirring at room temperature, the ethanol was evaporated to dryness and water was added. The mixture was stirred at room temperature for 30 min whereafter the product was precipitated with 2M hydrochloric acid.
Yield: 100%
.sup.1 H NMR (DMSO): 3.74 (8H, s); 4.27 (4H, s); 7.71 (2H, d, J=7 Hz); 7.87 (1H, t, J=8 Hz); 8.09 (2H, t, J=7 Hz); 8.33 (1H, d, J=8 Hz); 8.62 (1H, d, J=8 Hz); 8.63 (2H, d, J=7 Hz); 8.79 (2H, s); 8.83 (1H, s).
1-(2,6-Pyridyldicarbonylmethyl)-bispyridinium iodide, 13
2,6-Diacetylpyridine (8.16 g, 50.0 mmol) and iodide (25.38 g, 100.0 mmol) were heated to reflux in pyridine (125 ml) for 2 hours. The solution was cooled, and the brown crystalline pyridinium salt (13) separated by filtration.
Yield: 87%
6,6"-Dicarboxy-4,4"-diphenyl-2,2';6',2"-terpyridine, 16a
A solution of 13 (2.86 g, 5.0 mmol), 2-oxo-4-phenyl-3-butenoic acid (1.76 g, 10.0 mmol) and ammonium acetate (8.86 g, 115.0 mmol) in acetic acid (50 ml) was heated to reflux for 5 hours, whereupon the brown solution as cooled. The solid material was filtered and dried in air. The ammonium salt of 2 was dissolved in water and acidified with 6M HCl (pH about 1). The product was filtered and washed with water.
Yield: 34%
m.p. 279.degree. C. (dec.).
.sup.1 H NMR (DMSO): 7.56-8.06 (10H, m); 8.27 (1H, t); 8.40 (2H, d); 8.70 (2H, d); 9.18 (2H, d).
IR (KBr): 1760, 1715, 1695, 1420, 1365, 1272 cm.sup.-1 (C.dbd.O and C--O)
6,6"-Dicarboxy-4,4"-bis(4-nitrophenyl)-2,2';6',2"-terpyridine, 16b
Compound 16b was synthesized from 13 and 15b using a method analogous to the synthesis described in 16a.
Yield: 57%
.sup.1 H NMR (DMSO): 8.24 (1H, t); 8.28 (4H, d); 8.37 (4H, d); 8.43 (2H, d); 8.65 (2H, d); 9.13 (2H, d).
6,6"-Dicarboxy-4,4"-bis(3-nitrophenyl)-2,2';6'2"-terpyridine, 16c
Compound 16c was synthesized from 13 and 15c using a method analogous to the synthesis described in 16a.
Yield: 81%
6,6"-Bis(methoxycarbonyl)-4,4"-diphenyl-2,2';6'2"-terpyridine, 17a
One drop of concentrated sulfuric acid was added to a suspension of 16a (0.77 g, 1.6 mmol) in methanol (5 ml). The mixture was heated to reflux for 24 hours. The cold mixture was filtered ad the product was crystallized from dichloromethane.
Yield: 61%
m.p. 285.5.degree.-286.5.degree. C.
.sup.1 H NMR (CDCl.sub.3): 4.13 (6H, s); 7.49-7.88 (10H, m); 8.07 (1H, t); 8.44 (2H, d); 8.67 (2H, d); 9.07 (2H, d).
IR (KBr); 1741, 1725, 1255, 1144 cm.sup.-1 (C.dbd.O and C--O).
6,6"-Bis(methoxycarbonyl)-4,4"-bis(4-nitrophenyl)-2,2';6',2"-terpyridine, 17b
Compound 17b was synthesized from 16b using a method analogous to the synthesis described in 17a.
Yield: 45%
.sup.1 H NMR (CDCl.sub.3): 4.05 (6H, s); 7.80-9.03 (15H, m).
IR (KBr): 1740, 1250, 1125 cm.sup.-1 (C.dbd.O and C--O), 1525, 1345 cm.sup.-1 (NO.sub.2).
6,6"-Bis(methoxycarbonyl)-4,4"-bis(3-nitrophenyl)-2,2';6',2"-terpyridine, 17c
Compound 17c was synthesized from 16c using a method analogous to the synthesis described in 17a.
Yield: 38%
.sup.1 H NMR (CDCl.sub.3): 4.05 (3H, s); 4.09 (3H, s); 4.09 (3H, s); 7.75-8.97 (15H, m)
IR (KBr): 1725, 1260, 1130 cm.sup.-1 (C.dbd.O and C--O), 1530, 1340 cm.sup.-1 (NO.sub.2).
6,6"-Bis(hydroxymethyl)-4,4"-diphenyl-2,2';6',2"-terpyridine, 18a
Sodium borohydride (0.13 g, 3.5 mmol) was added to a suspension of 17a (0.40 g, 0.8 mmol) in absolute ethanol (10 ml). After stirring for 2 hours at room temperature the mixture was heated to reflux for 15 hours. The solution was evaporated in vacuo. A saturated solution of sodium hydrogen carbonate (5 ml) was added to the residue. After the solution was brought to the boil, water (15 ml) was added. The mixture was allowed to stand overnight in the cold. The precipitate was filtered, dried in air and finally crystallized from methanol.
Yield: 47%
m.p. 218.degree.-219.degree. C.
.sup.1 H NMR (DMSO): 4.75 (4H, s); 7.52-7.95 (10H, m); 7.86 (2H, d); 8.14 (1H, t); 8.49 (2H, d); 8.81 (2H, d).
6,6"-Bis(hydroxymethyl)-4,4"-bis(4-nitrophenyl)-2,2';6',2"-terpyridine, 18b
Compound 18b was synthesized from 17b using a method analogous to the synthesis described in 18a.
Yield: 40%
6,6"-Bis(hydroxymethyl)-4,4"-bis(3-nitrophenyl)-2,2';6',2"-terpyridine, 18c
Compound 18c was synthesized from 17c using a method analogous to the synthesis described in 18a.
Yield: 56%
.sup.1 H NMR (DMSO): 4.98 (4H, s); 7.86-9.13 (15H, m)
IR (KBr): 1530, 1350 cm.sup.-1 (NO.sub.2).
6,6"-Bis(bromomethyl)-4,4"-diphenyl-2,2';6',2"-terpyridine, 19a
A solution of phosphorous tribromide (0.12 g, 0.4 mmol) in chloroform (1 ml) was added to a solution of 18a (0.13 g, 0.3 mmol) in chloroform (17 ml). The mixture was refluxed for 11 hours whereafter the mixture was neutralized with 5% sodium hydrogen carbonate. The aqueous layer was extracted with chloroform (3.times.20 ml). The combined organic phase was dried with sodium sulfate and evaporated in vacuo. The residue was crystallized from dichloromethane.
Yield: 29%
m.p. 253.5.degree.-255.5.degree. C.
.sup.1 H NMR (CDCl.sub.3): 4.73 (4H, s); 7.47-7.81 (10H, m); 7.73 (2H, d); 8.01 (1H, t); 8.57 (2H, d); 8.79 (2H, d).
6,6"-Bis(bromomethyl)-4,4"-bis(4-nitrophenyl)-2,2'; 6',2"-terpyridine, 19b
Compound 19b was synthesized from 18b using a method analogous to the synthesis described in 19a.
Yield: 37%
.sub.1 H NMR (CDCl.sub.3): 4.75 (4H, s); 7.40-8.70 (15H, m).
6,6"-Bis(bromomethyl)-4,4"-bis(3-nitrophenyl)-2,2';6',2"-terpyridine, 19c
Compound 19c was synthesized from 18c using a method analogous to the synthesis described in 19a.
Yield: 50%
.sup.1 H NMR (CDCl.sub.3): 4.78 (2H, s); 4.83 (2H, s); 7.48-8.65 (15H, m)
6,6"-Bis[N,N-bis(carboxymethyl)aminomethyl]-4,4"-diphenyl-2,2';6',2"-terpyr idine, 20a
Potassium carbonate (120 mg, 0.9 mmol) was added to a mixture of 19a (47 mg, 0.08 mmol) and di-t-butyl iminodiacetate (41 mg, 0.20 mmol) in dry acetonitrile (5 ml), and the mixture was stirred for 24 hours at room temperature. The mixture was evaporated in vacuo, the residue was stirred with chloroform (10 ml), washed with water (2.times.10 ml) and dried with sodium sulfate. Evaporation gave an oil which was characterized by NMR spectrum. The oil was dissolved in trifluoroacetic acid (3 ml) and kept at room temperature for 16 hours. The trifluoroacetic acid was evaporated in vacuo, the solid residue was triturated with ethyl ether.
Yield: 59%
.sup.1 H NMR (DMSO): 3.71 (8H, s); 4.27 (4H, s); 7.54-7.96 (10H, m); 8-04 (2H, d); 8.18 (1H, t); 8.51 (2H, d); 8.85 (2H, d)
IR (KBr): 1734, 1630, 1395, 1200 cm.sup.-1 (C.dbd.O and C--O).
6,6"-Bis[N,N-bis(carboxymethyl)-aminomethyl]-4,4"-bis(4-aminophenyl)-2,2';6 ',2"-terpyridine, 20b
The reaction between 19b (0.11 g, 0.2 mmol) and diethyl iminodiacetate (76 mg, 0.40 mmol) in dry acetonitrile (10 ml) and potassium carbonate (0.27 g, 20 mmol) was made in analogy with 20a. The reaction gave an oil which was purified with flash chromatography (silica, MeOH/CHCl.sub.3 1/19). The oil was dissolved in methanol (5 ml). 10% Palladium on carbon (20 mg) was added followed by slow addition of sodium borohydride (23 mg, 0.60 mmol). After one hour the mixture was filtered and the filtrate was evaporated in vacuo. The residue was dissolved in 0.5M potassium hydroxide ethanol (20 ml). After stirring for 3 hours at room temperature the mixture was evaporated and water (20 ml) was added. The mixture was acidified with 6M HCl (pH about 2.0). The product was filtered and washed with water.
Yield: 60%
.sup.1 H NMR (DMSO): 3.71 (8H, s); 4.30 (4H, s); 7.75-8.80 (15H, m)
IR (KBr): 1735, 1630, 1400, 1200 cm.sup.-1 (C.dbd.O and C--O).
6,6"-Bis[N,N-bis(carboxymethyl)-aminomethyl]-4,4"-bis(3-aminophenyl)-2,2';6 ',2"-terpyridine, 20c
Compound 20c was synthesized from 19c using a method analogous to the synthesis described in 20b.
Yield: 79%
.sup.1 H NMR (DMSO): 3.68 (8H, s); 4.21 (4H, s); 7.63-8.64 (15H, m)
IR (KBr): 1725, 1630, 1400, 1190 cm.sup.-1 (C.dbd.O and C--O).
L-Lysine ethyl ester (starting compound, Scheme 3)
Thionyl chloride (5.0 ml, 8.06 g, 68 mmol) was added dropwise to 500 ml of ice- cooled dry ethanol. The stirred mixture was kept for 20 min at this temperature and L-lysine hydrochloride (20 g, 109 mmol) was added.
The mixture was then refluxed for 3 h and concentrated to a volume of about 200 ml. 200 ml of diethylether was added and the crystallized product filtered off.
Yield: 29 g (97%)-dihydrochloride. Rf=0.20 (System F)
.omega.-N-(4-Nitrobenzoyl)-L-lysine ethyl ester (37)
L-lysine HCl (5 g, 27.4 mmol) dissolved in 50 ml of water was titrated with 5M NaOH to pH 10.5. 4-Nitrobenzoyl chloride (6.6 g, 36 mmol) in dioxane (50 ml) and 5M NaOH were slowly added keeping the vigorously stirred reaction mixture at pH 10.5.
After complete addition and disappearance of the pink colour the reaction mixture was acidified with conc. HCl to pH 2 and extracted four times with diethylether. The aqueous phase was concentrated to dryness, coevaporated twice with 200 ml of dry ethanol and suspended in 250 ml of dry ethanol previously treated with 10 ml of thionyl chloride. The mixture was refluxed for 3 h, filtered and evaporated. The residual material was partitioned between saturated sodium bicarbonate and chloroform/ethanol 1:1 and the organic phase was dried over magnesium sulfate yielding a crude product which was purified by flash chromatography using 5% EtOH/chloroform as eluent.
Yield: 1.08 g (12%) oil crystallizing on standing Rf=0.23 (System A)
H.sup.1 NMR (60 MHz, CDCl.sub.3): 8.25 (d,2H, J=9 Hz), 7.93 (d, 2H, J=9 Hz), 6.87 (s, broad, 1H), 3.99-4.34 (q, 2H), 3.30-3.60 (m, 3H), 1.40-1.75 (m, 8H), 1.11-1.37 (t, 3H)
.alpha.-N-(Methoxycarbonylmethyl)-.omega.-N-(4-nitrobenzoyl)-L-lysine ethyl ester (38)
Compound (37) (0.54 g, 1.7 mmol) was coevaporated with toluene, dissolved in dry acetonitrile (10 ml) and bromoacetic acid methylester (0.265 g, 1.7 mmol) was added followed by pulverized dry sodium carbonate (2.0 g). The mixture was refluxed for 3 h.
Filtration of the inorganic salts and evaporation of the acetonitrile gave an oily crude product which was purified by flash chromatography.
Yield=0.45 g (68%) oil Rf=0.26 (System A)
H.sup.1 NMR (60 MHz, CDCl.sub.3): 8.25 (d, 2H, J=9 Hz), 7.93 (d, 2H, J=9 Hz), 6.63 (s, broad, 1H), 3.95-4.30 (q, 2H), 3.68 (s, 3H), 3.30-3.60 (m, SH), 1.40-1.75 (m, 7H), 1.11-1.37 (t, 3H).
.omega.-N-Monomethoxytrityl-L-lysine ethyl ester (39)
Dry triethylamine (1.8 ml, 18 mmol) was added to a suspension of the starting compound (scheme 3) (1.5 g, 6 mmol) in 20 ml of dry pyridine. To this mixture stirred at RT, solid monomethoxytrityl chloride (1.96 g, 6 mmol) (MMTrCl) was added in small portions during a period of 1 h whereupon the mixture was stirred for additional 2 h. A standard sodium bicarbonate work-up, followed by extraction with chloroform, yielded a crude product contaminated with .alpha.-MMTr isomer.
The pure title product was easily isolated by flash column chromatography due to the large Rf difference between the isomers.
Yield: 1.35 g (48%) oil Rf=0.43 (System A)
H.sup.1 NMR (400 MHz, CDCl.sub.3): 7.5-6.75 (m, 14H), 4.18-4.13 (q, 2H), 3.78 (s, 3H), 3.45-3.37 (m, 1H), 2.14-2.10 (t, 2H, J=7 Hz), 1.75-1.35 (m, 9H), 1.26 (t, 3H)
.alpha.-N-(Methoxycarbonylmethyl)-.omega.-N-monomethoxytrityl-L-lysine ethyl ester (40)
A partially protected L-lysine derivative (39) (1.0 g, 2.13 mmol) was converted to product (40) using the method described in Example 36.
Yield: 0.81 g (70%) oil Rf=0.73 (System A)
H.sup.1 NMR (400 MHz, CDCl.sub.3): 7.46-6.77 (m, 14H), 4.19-4.14 (q, 2H), 3.77 (s, 3H), 3.70 (s, 3H), 3.31-3.45 (q, 2H), 3.22-3.25 (t, 1H), 2.09-2.12 (t, 2H), 1.35-1.70 (m, 6H), 1.23-1.27 (t, 3H)
.omega.-N-Trifluoroacetyl-L-lysine ethyl ester (41)
The starting compound (scheme 3) (2.0 g, 8.1 mmol) dissolved in 10 ml of dry ethanol was treated with dry triethylamine (4.09 g, 40.4 mmol). Ethyl trifluoroacetate (1.5 g, 10.5 mmol) was added to the stirred suspension formed, and the mixture was refluxed for 6 h.
All volatiles were then evaporated and the residue was partitioned between saturated sodium hydrogen carbonate and chloroform/ethanol 1:1.
The combined organic phase (5.times.60 ml) was evaporated, coevaporated with toluene and flash chromatographed to give the title product in the form of a colorless oil.
Yield: 1.9 g (87%) Rf=0.72 (System D)
H.sup.1 NMR (400 MHz, CDCl.sub.3): 7.10 (t, 1H, exchangeable), 4.21-4.16 (q, 2H), 3.45-3.40 (m, 1H), 3.38-3.31 (m, 2H), 1.84 (s, 2H, exchangeable), 1.82-1.40 (m, 6H), 1.28 (t, 3H).
.alpha.-N-(Methoxycarbonylmethyl)-.omega.-N-trifluoroacetyl-L-lysine ethyl ester (42)
L-lysine derivative (41) (1.0 g, 3.7 mmol) was converted to the product (42) by means of a method analogous to Example 36.
Yield: 1.05 g (83%) oil Rf=0.48 (System A)
H.sup.1 NMR (60 HMz, CDCl.sub.3 +CD.sub.3 OD): 4.4-4.0 (q, 2H), 3.68 (s, 3H), 3.5-3.1 (m, 5H), 1.8-1.4 (m, 6H), 1.23 (t, 3H).
.omega.-N-(4-Hydroxybutyryl)-L-lysine ethyl ester (43)
L-lysine etyl ester.times.2 NCl (36) (2 g, 8.1 mmol) in 30 ml of dry ethanol was treated with dry triethylamine (5.63 ml, 40.5 mmol) and .gamma.-butyrolactone (0.7 g, 8.1 mmol) and the resultant suspension was refluxed for 3 h.
Evaporation of volatiles and coevaporation with toluene yielded a crude product which was purified by flash chromatography using 20% methanol/chloroform as solvent.
Yield: 1.54 g (73%) oil Rf=0.28 (System D)
1 H.sup.1 NMR (400 MHz, CDCl.sub.3 +CD.sub.3 OD): 4.30-4.22 (q, 2H), 3.72-3.77 (m, 1H), 3.58-3.65 (t, 2H), 3.18-3.28 (m, 2H), 2.30-2.36 (t, 2H), 1.40-2.00 (m, 8H), 1.28-1.34 (t, 3H).
Fluorescence of europium and terbium chelates of compound 20a
The relative fluorescence yield .phi..sub.rel of the europium and terbium chelates of compound 20a were measured in equimolar 10.sup.-5 M solutions of compound 20a and the corresponding lanthanide ion. Fluorescence measurements were done on a Perkin-Elmer LS-5.RTM. spectrofluorometer using the phosphorescence mode which allowed the decay curves of the lanthanide fluorescence to be measured. The fluorescence yield is reported relative to the fluorescence of the uncomplexed lanthanide cation (Ln) using the equation: ##EQU1## where I.sub.che and I.sub.Ln are the preexponential terms of the emission decay curves for the chelated and uncomplexed lanthanide cation, respectively (614 nm for europium and 544 nm for terbium). The excitation wavelength for the uncomplexed europium was 395 nm and for terbium 370 nm. C.sub.Ln and C.sub.che are the concentrations of free and complexed lanthanide cation, respectively, and k.sub.Ln and k.sub.che the corresponding decay constants. For compound 20a the relative fluorescence yield for the europium comples becomes 2.1.times.10.sup.6 and for the terbium complex 8.1.times.10.sup.4. The excitation wavelength for the chelates was in both cases 340 nm.
1. A lanthanide chelate in which a lanthanide selected from the group consisting of Eu.sup.3+, Tb.sup.3+, Sm.sup.3+ and Dy.sup.3+ is chelated to a terpyridine compound having the structure ##STR4## wherein (i) n=1 or 2,
- (ii) X.sub.1 =X.sub.5 hydrogen,
- (iii) X.sub.2, X.sub.3 and X.sub.4 are selected from the group consisting of hydrogen, hydroxy, amino, amido, alkoxy, alkyl, alkynyl, aryl, aralkyl, aryloxy, arylamino and arylethynyl, with the proviso that alkyl has less than 12 carbon atoms and aryl is selected from phenyl, quinolyl, naphthyl, pyridyl and pyrimidyl,
- (iv) Ch is a chelating group selected from --COO.sup.-, --OPO.sub.3.sup.2- and- PO.sub.3.sup.2-,
- (v) X-Y is a substituent replacing a group X.sub.1 -X.sub.5 or H', in which X is a bridge hot participating in the chelating and consisting of a pure hydrocarbon chain of 1-12 carbon atoms and at least one structural element selected from the group consisting of secondary and tertiary amines, aliphatic disulfides, aliphatic thioethers, ethers, esters and diaza, and
- Y is a residue of a compound selected from the group consisting of antigens, haptens, antibodies and nucleic acids, said residue retaining the biospecific affinity of said compound.
2. A chelate according to claim 1 wherein the pure hydrocarbon chain is selected from the group consisting of phenyl and naphthyl.
3. A chelate according to claim 1 wherein n=1; X.sub.2 and X.sub.4 are hydrogens; Ch is --COO.sup.-; X-Y is 4-aminophenyl to which the residue Y is linked at the 4-amino group; and X-Y replaces X.sub.3.
4. A chelate according to claim 1 wherein n=1; X.sub.2 and X.sub.4 are hydrogens; Ch is --COO.sup.-; X-Y is 3-aminophenyl to which the residue Y is linked at the 3-amino group; and X-Y replaces X.sub.3.
5. A chelate according to claim 1 wherein n=2; X.sub.3 is hydrogen; Ch is --COO.sup.-; X-Y is 4-aminophenyl to which the residue Y is linked at the 4-amino group; and X-Y replaces X.sub.2 and X.sub.4, respectively.
6. A chelate according to claim 2 wherein n=2; X.sub.3 is hydrogen; Ch is --COO.sup.-; X-Y is 3-aminophenyl to which the residue Y is linked at the 3-amino group; and X-Y replaces X.sub.2 and X.sub.4, respectively.
7. A chelate according to claim 2 wherein n=1; X.sub.2 and X.sub.4 are hydrogens; X-Y is isothiocyanatophenyl to which the residue Y is linked at the isothiocyanato group; and X-Y replaces X.sub.3.
Referenced Cited
U.S. Patent Documents
|January 20, 1987
|Hinshaw et al.
|November 15, 1988
|Schaeffer et al.
|January 31, 1989
|Burdick et al.
|June 6, 1989
|August 22, 1989
Foreign Patent Documents
|September 1986
|November 1986
Other references
- CA98(13):107123t Constable et al. Polyhedron 1(3):303-6, 1982. Mears et al. (1984) Anal. Biochem. 142;68-78.
Patent History
Type: Grant
Filed: Apr 19, 1991
Date of Patent: Apr 13, 1993
Assignee: Wallac Oy
Inventors: Jouko Kankare (Turku), Harri Takalo (Turku), Elina Hanninen (Turku), Matti Helenius (Turku), Veli-Matti Mukkala (Kaarina)
Primary Examiner: Christine M. Nucker
Assistant Examiner: Kay Kim
Attorney: Fred Philpitt
Application Number: 7/687,498
International Classification: C07K 1528; C07K 1702; A61K 3944; A61K 4902;


text:
Get Funagain Points by submitting media! Full details, including content license, are available here.
You must be logged in to your account to submit media. Please click here to log in or create a free account.
Home > Strategy Games >
The Resistance: Coup: Reformation
Notify me if/when this item becomes available:
(you will be asked to log in first)
Please Login to use shopping lists.
Product Description
Coup: Reformation, an expansion for the original version of Coup: City State from La Mame Games, adds new cards to the game and rules for factions and team play that increases tension in the early stages for four or more players and (thanks to 15 additional character cards) allows Coup to be better played with up to ten players.
With Coup: Reformation, each player must declare himself either Protestant or Catholic and can target only members of the other faith. Conversion is possible, however, for yourself or for another player by paying a charitable donation to the Almshouse. Like all factions, once you have eliminated the other sect, you just descend into in-fighting, so there's still only one winner and no second place.
Coup: Reformation adds a new fluid team dynamic to Coup as players jostle with their allegiance to take advantage or seek protection in the early stages of the game.
Product Information
Indie Boards & Cards
Year: 2014
Players: 4 - 10
Time: 15 or more minutes
Ages: 10 and up
Weight: 106 grams
Product Reviews
Average Rating: 2 in 1 review
The cards arrived with many errors. Some cards aren't the same when they should be. I'm disappointed by the poor production. Some cards are simply not usable.
For example, the game comes with 10 instruction cards. But 4 of the instruction cards are completely different than the other 6.
I would recommend waiting for next edition/reprint before buying.

subset: commoncrawl


text:
Landon Collins' Key to Success
When you talk to Landon Collins, it's clear why he's one of the best safeties in the NFL. He had a breakout season last year with 125 tackles, four sacks, 13 pass deflections, five interceptions and a touchdown in 16 starts, and he's on track for another incredible year. He's only 23 years old and what Landon has already accomplished is extraordinary.
Landon's biggest key to success: practice. The University of Alabama alum developed a strong work ethic playing under Head Football Coach Nick Saban and today he treats every practice with the same intensity as a real game.
We all want to be great at what we do, but not all of us are willing to practice our craft over and over and over again. In Landon's case, it's the commitment to preparation and practice that makes the difference between ordinary and extraordinary.


text:
Trick or Treat Slot Game Review
Play the Trick or Treat free slots if you are looking for games with eye-catching graphics, plenty of features and a big jackpot prize. It is a 5 reel video slots with a wild, scatter and free spin.
Each slot reel is pulsating with a rising green mist that is eerie and a scary-looking haunted house surrounds the entire gaming area.
The game is designed with nice touches which include creepy trees on both sides of the slot reels as well as line markers in shapes such as tasty treats.
The reel icons have an ultra-modern look with some including cutting-edge animations. Of course, all the icons are themed around trick or treating and Halloween.
These include things like giant lollypops, eggs, devils horns, fake blood, candy bags, witches hats, pumpkins, wolf masks and children dressed up as ghouls and ghosts.
Features of the Trick or Treat Free Slots
There is the pumpkin or jack o lantern icon that can substitute for others except for the scary child. When five, four or three icons show up, the scary child cloak wearing icon will give 20, 15 or 10 free spins. They can show up in any position to trigger the spins which will begin immediately. Your winnings will be doubled in the free spins to give you as much as 12,000 coins during each spin.
A gamble feature is present as well and this is themed on the actual trick or treating act. Following each win, you can choose the gamble button that will load a different screen with an eerie door in the middle. You will either choose from 4 suits to quadruple it or pick the next color suits to double your winnings.
When you decide to play, you should choose a suitable bet to spin the slot reels using automatic spins or single spins.
Overall, the Trick or Treat Slots is a great game for anyone who loves Halloween and everything that is related to it. When playing, you will be treated to some stunning graphics as well as many other exciting features that can enhance your experience.


text:
If you've had An on the job exposure, call us 24/7
(Must be a WellAmerica Client)
TB (tuberculosis) skin testing for public safety
After years of decline TB is back and on the increase. Pima County has had the highest rates in the state.
Skin test:
10 mm or more of induration (swelling) is positive for health care and public safety workers with normal immunity. Redness alone doesn't count. If positive the doctor/nurse practitioner will decide:
Need for initial chest x-ray (may have just had one elsewhere so no need to repeat).
Radiologist may request extra x-ray views if any concerns on initial x-ray.
If your Chest x-ray is negative you do not have active TB
You can't give it to anyone at home or work
Because ten percent of people with a positive skin test and NEGATIVE chest x-rays will develop active TB later in life there's a medication to prevent that.
TB prophylaxis (medication):
Risks of medication to the liver increase with age, especially after age 35.
Usual medication is INH (isoniazid) 300 mg a day for 9 months
If you are placed on medication have your liver functions (blood test) tested periodically.
Where to go for help:
See your personal physician.
Pima County Health Department – Note, there is a charge.
If positive skin test is from an exposure, then see (1) or (2) or your companies/organizations occupational medical clinic.
Who pays:
Your agency covers cost of TB skin tests.
If you need a chest x-ray or medication you would have to prove to your worker's compensation carrier that you became ill at work for it to be considered an industrial case.
Some agencies will pay cost of the above.
Most do not and advise you to seek care at your personal physician
Symptoms of TB:
Chronic cough with phlegm, often bloody.
Night sweats, fever, weight loss.
Further information: CDC.org., Pima County Health Department, Tuberculosis.
Bed Bugs at a Glance
A downloadable PDF: Bed_Bugs_At_A_Glance_WellAmerica
Bed bugs are small, oval non-flying insects that feed by sucking blood from humans or animals. Bed bugs can live in any area of the home and can reside in tiny cracks in furniture as well as on textiles and upholstered furniture. They tend to be most common in areas where people sleep and generally concentrate in beds, including mattresses, box springs, and bed frames.
Bed bugs are most active at night and bite any exposed areas of skin while an individual is sleeping. The face, neck, hands, and arms are common sites for bed bug bites. A bed bug bite is painless and is not noticed. Small, flat, or raised bumps on the skin are the most common sign: redness, swelling, and itching commonly occur.
Typically, no treatment is required for bed bug bites. If itching is severe, steroid creams or oral antihistamines may be used for symptom relief. Fecal stains, egg cases, and exuviae (shed skins) of bed bugs in crevices and cracks on or near beds are suggestive that bed bugs may be present, but only observing the bugs themselves can confirm an active infestation.
A professional pest-control company may be required to help identify and remove bed bugs from the home or workplace.
Prevention of bed bug bites?
Avoidance of infested areas is the method for prevention of bed bug bites. Recognition of bed bug infestation and proper treatment of affected rooms (usually with the help of a pest-control specialist) is the best way to prevent bed bugs in the home. Those concerned about the potential for bed bugs bites in hotels, dorms, or other common sleeping areas (fire house, etc.) should examine beds and mattresses for signs of a bed bug infestation. Sealing your mattress in a bed bug prevention casing can be beneficial.
Getting Rid of Bed Bugs
Getting rid of bed bugs is not an easy process, and most cases of bed bug infestation will require treatment by a pest-control expert. A variety of low-odor sprays, dusts, and aerosol insecticides can be used to eradicate bed bugs. These must be applied to all areas where the bugs are observed as well as spaces where they may crawl or hide. The pest-control company can help you determine if the mattress can be disinfected or must be discarded. Since beds cannot readily be treated with insecticides, it's often necessary to discard infested mattresses and beds.
The pest-control expert may recommend certain forms of deep-cleaning such as scrubbing infested surfaces with a stiff brush to remove eggs, dismantling bed frames and furniture, filling cracks in floors, walls, and moldings, encasing mattresses within special bags, or using a powerful vacuum on cracks and crevices.
How do I get rid of bed bugs?
Eradication of bedbugs frequently requires a combination of pesticide and non-pesticide approaches. Pesticides that have historically been found to be effective include: pyrethroids, dichlorvos and malathion. Resistance to pesticides has increased significantly over time and there are concerns of negative health effects from their use. Mechanical approaches such as vacuuming up the insects and heat treating or wrapping mattresses have been recommended.
The carbamate insecticide propoxur is highly toxic to bedbugs, but the Environmental Protection Agency (EPA) has been reluctant to approve such an indoor use because of its potential toxicity to children after chronic exposure.
University Medical Center Bug-O-Gram, 8/2011
Greenberg, L., and J. H. Klotz. "Pest Notes: Bed Bugs." Oakland: Univ. Calif. Nat. Agric. Res. Publ. 7454. Sept. 2002.
Harvard School of Public Health
Potter, Michael F. "Bed Bugs." University of Kentucky College of Agriculture. Aug. 2008.
Thomas, I., G.G. Kihiczak, and R.A. Schwartz. "Bedbug Bites: A Review." Int J Dermatol 43 (2004): 430.


text:
Guy Yanai
Born in 1977, Haifa, IL
Lives and works in Tel Aviv, IL
M.F.A., Hampshire College, Amherst, MA, US
Pont-Aven School of Art, Pont-Aven, FR (summer-term)
B.F.A., Parsons School of Design, New York, NY, US
Guy Yanai's paintings are characterized by bold colors, simplified shapes, and a flattened depth of field. In his work, the banal is reduced to geometric segments in a stripping away of references to the tangible world in favor of a visual experience that is more akin to digital imagery—using Google Street view as a reference for a landscape for example. He often chooses everyday objects and spaces as his subjects, flattening and abstracting them in a way that seems removed and objective. Yanai has cited numerous sources of inspiration—from old, modern, and contemporary masters to digital imagery, television and advertisements—and his simplified representations may serve as a way to condense and organize the multitude of data we receive in reference to any person, place, or thing into concise representations with tight compositions. The more time spent with Yanai's painting, the more it encourages an investigation into the perceptive space of the canvas, a place where objects, scenery and nature are conjured, sustained and gently teased away.
Los Angeles
For inquiries regarding Guy Yanai, please complete this form:


text:
A mother to anotherRead Now
For those of us that travel via plane (frequently or not), it is not uncommon to still be flabbergasted that just a short time ago, we were literally on the other side of the world! The technology that has allowed us to span the earth has essentially now made the phrase "what a small world" so true.
Last week, I was in Haiti and the smiles, successes and sweet stories are still so fresh - I catch myself pondering how I was just there!
We were on our way to a family's house where we got the opportunity to build a roof. This family of 8 had been living in a tarp tent since the 2010 earthquake. Another group had helped build the walls to the house a few years prior, but they still lacked the resources or partnership to put the final piece of the structure together. Ten students from Valor Christian School International, along with an equal number of students from the Maranatha House, made their way along the dusty streets to the house.
I was bringing up the rear, and the site in front of me brought tears to my eyes. Tears of joy that is.
For the past 7 years, as I've gotten to know the children and young adults who have lived at the Maranatha House Orphanage, I have seen the effects poverty and broken families have played. To be a child of an orphanage in Haiti, you are there because either your parents are no longer alive or are unable to care for you in the ways you need. For most of the living parents, sending a child to an orphanage is an act of desperate love! While they believe they are giving their child the best opportunity (and in many cases they are), the lack of physical, tangible, communal love with their parents has a deep impact on them.
Some of the children still have living family members visit them - and we know this is a valuable and important part of their story. We never wish to take that away, and if ever possible, our goal is for reunification of children with their families. However, we know that for the youth we serve to truly be successful, their need for consistent parental love and support is essential. It is for this reason that we are continually motivated to provide the best services and people support for these incredible young people.
What I saw last week, walking in front of me, was a true example of love and support for the young people of Haiti. I saw a mother's love.
Kekette was linked arm in arm with one of the young women in our Transitions program, and one from the Maranatha House.
Kekette is our female Transitions Coordinator. Her role consists of working with the older youth who have transitioned out of life in the orphanage to life on their own. She is there to guide, support and hold them accountable to their goals and dreams. Additionally, she works with the youth still living at the orphanage, helping to prepare them for the inevitable transition into adulthood. This could be just a job for Kekette, but for her it is SO MUCH MORE.
As a mother of 11, Kekette knows a thing or two about raising children. Many of her own children have gone on to have successful careers, are in college, and are following their dreams. She also worked for a number of years doing this same work for another organization. It goes without saying that she is qualified for the position.
But what makes her extraordinary, is the way she cares for each young person she encounters. She loves them as if they were her own. She sees in each of them their unique God-given purpose. She wants the BEST for the youth she works with, and works long hours and has long conversations in order to push each of them to do their best, be their best, and pursue their best.
While my Creole is still rusty, I could clearly understand the LOVE that was clearly understood in the way that she and the youth spoke with each other. She can be tough and have high standards, but it was evident that her love for them was never questioned.
Kekette, along with our male Transitions Coordinator, Achemetre, are essential to the overall success of this program and it's ability to support youth as they transition from orphanage life to a THRIVING adult life.
When you sponsor a young person in our Transitions Program, you are providing exceptional role models and influencers like Kekette for them. Your sponsorship is giving them the guidance and the love that they need to be their best selves and face adulthood with courage. Through Kekette and the rest of the team, you are meeting this deep need to be known and loved. To be valued and supported.
Traveling to Haiti isn't the only way to impact the future there!
Become a sponsor today!
When fully sponsored, our Transitions Program will provide each youth with:
Each one of our youth in the Transitions Program needs 7 sponsors providing $35/mo to provide them with all of these essential services and support.
Sponsors are radically impacting the future of Haiti by investing in the lives of an individual - an individual who has the God-given potential to be the leader their community so desperately needs!
Please consider becoming a sponsor today!
Katie Matheny
Ncompass is striving to become a leader in impacting and building up today's youth for the Kingdom of God. We are committed to taking a bold, innovative and strategic business approach to inspire global action.
August 2019


text:
MIAMI BEACH — This week, the Art Basel Miami Beach and Design Miami fairs have brought thousands of art aficionados to Florida. But they will have to look beyond the usual venues to see one prominent new artwork: a parking garage by the Swiss architecture firm Herzog & de Meuron.
The building, known as 1111 Lincoln Road, features seven exposed concrete slabs supported — seemingly precariously — on tilted concrete columns. It rises 125 feet from a row of ground-floor storefronts to a 5,200-square-foot penthouse with a sloping terrace.
Robert Wennett, who developed the building, says he believes its striking geometry gives it the power of minimalist art. "The idea," Mr. Wennett said, "was it should not look or feel like a garage."
With a prestigious architectural pedigree, it joins garages by the likes of Frank Lloyd Wright, Paul Rudolph and the Miami firm Arquitectonica, which are included in "Houses of Cars: Innovation and the Parking Garage," a show at the National Building Museum in Washington (through July 11).
Terence Riley, an architect who until October was the director of the Miami Art Museum, said Mr. Wennett's building was also "a chance to show Miami Beach that important architecture doesn't have to be Art Deco."Continue reading the main story
Still, for all its architectural bravado, the real artistry in the building may be the deal-making that made it happen. When Mr. Wennett bought the site, which included an office building, in 2005, zoning regulations would have permitted him to construct about 50,000 square feet. But given the shortage of parking in Miami Beach, the city doesn't count most parking spaces in a building's square footage for zoning purposes.
This meant that by adding parking, Mr. Wennett was able to significantly increase the square footage he could build. And with that additional room, he could create an imposing structure that would attract high-end tenants to its 13 retail and four restaurant spaces.
So far, all but one of the retail spaces are rented, he said, at annual rents of up to $150 a square foot, which is high for Miami Beach. The first restaurant to sign on, which will open early next year, is a Shake Shack — an outpost of the Manhattan burger phenomenon and the first venture outside New York City by Danny Meyer's Union Square Hospitality Group.
Retail tenants include Taschen, the German book company, and Nespresso, the cult coffee marketer. The one space that isn't at ground level — a glass box on the fifth floor of the garage — is being taken by Alchemist, a boutique featuring designers Rick Owens and Martin Margiela and the jeweler Chrome Hearts. Alchemist already has a Lincoln Road location, but Roma Cohen, who owns the company with his wife, Erika, said that they "fell in love with the space, and there was no way we would have let someone else take it. When will this ever come up again?"
But one potential tenant withdrew from the project this fall: Genius Jones, a high-end children's "design store" with three other locations in South Florida, had reserved a space but decided not to occupy it. In the last year, "business on South Beach has declined significantly," the company's owner, Daniel Kron, said. "So it's just not the right moment for us to open an expensive new store in South Beach."
"Most of the other retailers coming to 1111 are big international companies looking for their first toehold in Miami," Mr. Kron said. "For them, it's a different story."
Mr. Wennett made his pitch directly to those companies' top executives, stressing the power of the architecture to attract and excite customers. "Product can be sold much better on the Internet," he said. "The reason to take physical spaces is to create an experience."
Mr. Wennett, 49, has spent 25 years in commercial real estate, mostly in the Northeast. In 1998, he and a partner created what became Starwood Urban Investments, based in Washington. By 2004, the fund owned some $500 million in properties in urban centers, and it decided to liquidate all of its holdings. That, Mr. Wennett said, "was very good timing."
The sale left him a lot of money, and the desire, he said, for a solo project. In the meantime, he had moved to Miami, where he became interested in the SunTrust Building, a concrete office tower from the 1960s. It anchored the western entrance to the Lincoln Road Mall — a pedestrian promenade designed by Morris Lapidus in 1960 and extensively renovated in the 1990s.
The building held about 100,000 square feet of offices; the lot next door contained a bank branch and some 50 parking spaces.
Mr. Wennett was able to buy the combined property, he said, for $23.5 million from a German company, and he began making plans to upgrade the existing structure and build on the adjacent lot, adding ground-floor storefronts in both parcels.
He considered 10 architects, he said, before settling on Pierre Herzog and Jacques de Meuron, who designed the "bird's nest" stadium at the Beijing Olympics and who, he said, were fascinated by the chance to build in a city as culturally rich as Miami Beach. (They have since designed the new home of the Miami Art Museum; it has yet to break ground.)
The Pritzker Prize-winning architects are known for the exquisite skins of their buildings — including the perforated copper sheets of the de Young Museum in San Francisco and the curved "bubble" glass of their Prada Tokyo store.
But 1111 Lincoln Road has no skin. According to Jacques Herzog, it's pure Miami Beach: "All muscle without clothing."
Creating a structure 15 feet taller than the SunTrust Building required approvals from the city's Design Review Board and Board of Adjustment, which Mr. Wennett received the first time he presented the plans.
Once the building was approved, Mr. Wennett turned to a related project — transforming the street in front of the building into an extension of the Lincoln Road Mall. He paid to have Raymond Jungles, a prominent Miami-based landscape designer, sketch designs for an "urban glade," inspired in part by the work of Mr. Lapidus. The city has also approved those plans, and construction is under way.
Mr. Wennett said he spent about $8 million out of his own pocket on the design and approval phases of the project.
This meant that, by the time he sought a loan for the project, in 2007, most of the risk had been eliminated and "it was easy to find financing." Ultimately, he said, he borrowed about $30 million of the project's construction costs, which he estimates at $57 million. He said he owned 93 percent of the partnership that controls the property; two limited partners hold the rest.
The completed complex includes a new two-story building with a SunTrust Bank branch on the ground floor and four large condos on the second. Mr. Wennett is selling the four condos — they are listed for about $2.5 million each — but will otherwise be dependent on rent to make back his investment. He said the garage's 300 parking spaces will go for market rates — no architecture premium.
He said he had benefited from the dip in the economy, because when he broke ground in 2007, "construction costs were probably 20 percent less than they would have been at the top of the market." Also, he said, he got his choice of contractors.
And, he said, it helped a lot that he was doing an architecturally distinguished building, which attracted tenants even in tough times. "If we had built a normal project," he said, "it probably never would have happened."Continue reading the main story


text:
by Jarrett A. Lobell (Archaeology magazine)
Early last year, archaeologists working on the route of a controversial highway near the village of Lismullin, Ireland, stumbled across a vast Iron Age ceremonial enclosure, or henge, surrounded by two concentric walls. The 2,000-year-old site is just over a mile from the Hill of Tara, traditional seat of the ancient Irish kings and site of St. Patrick's conversion of the Irish to Christianity in the fifth century A.D. The discovery of the massive henge, measuring more than 260 feet in diameter, confirms the long-held belief that the area around the hill contains a rich complex of monuments.
The extraordinary amount of archaeological remains on the Hill of Tara — burial mounds, religious enclosures, stone structures, and rock art dating from the third millennium B.C. to the twelfth century A.D. — makes it Ireland's most spiritually and archaeologically significant site. Construction of the new M3 highway, meant to ease traffic congestion around Dublin, threatens not only the Hill of Tara's timeless quality, but also newly discovered archaeological sites in the surrounding valley.


text:
My Love Dresses
Game info
57 plays
Date added: 2012-01-16
Description: In Mina's dressings, always keep many new clothes there. Huh, she is a dressing addict! She wanna keep her new outfile everyday and be the fashion everytime. Give her a lovely dressing now!
Tags: dressup makeup girlsgames.
Log-in to add a comment
More games


text:
Smart Ideas: Tips Revisited
Basic Information on Online Fitness Challenges Everything has been made so much easier because of technology so be sure to take full advantage of it. You can find convenience when it comes to these matters so be sure to take advantage of it. You always have to consider your health when it comes to the state of your everyday life. You need to consider the gym when it comes to this matter because it's what other successful people are taking advantage of as well. You would have everything you need and more by taking advantage of the online gym. You can accomplish your objective through online fitness challenges. You just have to use the internet and you'd be okay. You can learn everything from your own home and develop self-discipline at the same time. Technology would surely provide you with all you could ever require regarding this venture. Taking care of your system has never been this easy before. Find the perfect exercise for you by doing some research in your end. There are all kinds of moves that would make you happy to have tried out. Online assistance is great when it can give you these amazing gifts. You could only imagine how this venture would become so much more affordable when you are smart about your decisions in the first place. When you make use of these methods, you can improve the state of your health as you please. This is all about rising to the challenge and achieving your dreams at the same time. Constant training is what you need to get fit again. A good routine would be ideal but make sure to change things up every once in a while. Boost the results in your body with the ideal online programs. These kinds of matters can be solved easily when you read this article from start to finish. There are so many ways in which people will savor challenges especially those that want to make a change. These challenges are things that other people would be willing to engage in with you as well. This can truly offer them with a ton of convenience so you basically have nothing to worry about. You would require the help of the online world to continue challenging yourself and others. With this kind of help, there is no reason you would ever feel bored again. You can even engage in these matters with other people who are willing to do it. Choose online fitness challenges that will turn your body into something you have always dreamed of. You need to open your eyes when it comes to matters you truly need. You will truly have fun and look exceptional as well because of this.What I Can Teach You About Resources


text:
About Stephan
Stephan Hoglund was born in Parkville, Minnesota, and grew up on the hard rock hills of the Laurentian Divide, where waters part on northern Minnesota's Iron Range.
To explain his work one must say he is very much a regionalist. The work comes from a sense of place or shall we say places. Northern Minnesota is where it first began and now the open High Mesa and High Desert of Southwest New Mexico near Silver City. These places not only have been the source of the materials in much of the work, but also seem to sustain a creative and spiritual roadmap. The shores of Lake Superior has been a major source of surface found Lake Superior Agate, Isle Royal Greenstone or Chlorastrolite and the the amazing and rare Lake Superior Thomsonite. It has been said It is if he was chosen to be a messenger for these Gems. Over the years he has collected, arguably the most extensive collection of Lake Superior Gemstones in the world, enough to share with you for the rest of his life. Lake Superior Gemstones and the many varieties of Turquoise and Variscite of the Southwest are some of the most valuable surface found gemstones in the world and are naturally the most environmentally sustainable and green gemstones in the world. For a true Minnesota Lake Superior Region or South West New Mexico treasure see his work and decide for yourself!
Stephan is one of the better known Minnesota Art Jewelers and was awarded a McKnight Fellowship for his Art Jewelry featuring the rare Gemstones of this region. He has one of the oldest and better known Minnesota Jewelry Design Studios in the region having been a mainstream presence for nearly 40 years. He creates Art and Studio Jewelry as well as wedding rings from Isle Royale Greenstone (Chlorastrolite) that depict his Boreal and Desert Environment. He also works with traditional stones such as Diamond Sapphire and Ruby.


text:
Foreign Policy Lessons from the Private Sector: Five Minutes with Senator Chuck Hagel
The Journal sat down with Senator Chuck Hagel to ask him a few questions about how his experience in the public and private sector has influenced his foreign policy views and for his analysis of some pressing geopolitical issues. View the rest of the interview, which details his perspective on American foreign policy and defense issues, as it appeared in issue 13.1 "Language, Identity & Politics" here.
GJIA: Senator Hagel, you co-founded Vanguard Cellular and were also president of the McCarthy Group. How did a career in the private sector prepare you for your time as a United States senator?
Senator Hagel: Well, I think that any experience in business is helpful because it rounds out your perspective and understanding of the world. Our world is now interconnected in every way. You cannot separate an examination of economic development from one regarding energy, resources, water, geopolitical relationships, type of government, or education. For me, business was helpful because it gave me not just a conceptual understanding of economies and relationships, but also the hands-on experience of meeting with world and business leaders. I can see, because of my own practical experience, how institutions like the G-20 fit into the dimensions of American sovereignty and government.
There is a very significant political tension in this country regarding China. The passage of the recent currency bill in the Senate and the public's blaming China for taking our jobs are two examples of this friction. Despite this, we need to cooperate and find geopolitical relationships built on common interests. We can work off of those common interests to deal with the differences. So for me, my experience in the private sector helped me to put all of these pieces and components together. It's also important politically too, because when you talk to your constituents about foreign policy, most Midwesterners don't focus on so called "university" foreign policy. Foreign policy to them means more about open markets in which they can sell their products. So a relationship with China and India, or any other country for that matter, is important because it represents more opportunities for them to sell their grain, beef, soybeans, and so on. That is a real life intersection where economy, business, government, foreign relations, and national interests come together. And that national interest is certainly economic as much as anything else.
GJIA: Senator, you are a director of the board of Chevron. Chevron works within a number of countries with authoritarian regimes, such as those in place in Angola, Saudi Arabia, Russia, and Venezuela. In your capacity as a director of the board, how do you ensure that human rights and corporate social responsibility are a priority for your company?
Senator Hagel: Well, that is always an issue. It has to be an issue for any corporation. You have to factor in all of the pieces: the interests of your company, country, people, and host country's people. You try to balance everything the best you can with the influence you have. How can you best influence the outcomes? Many corporations, whether it's Chevron, an oil company, or General Motors, have been doing business in China for 20 or 30 years. China is a communist country; so is Vietnam. Are you not going to do business with them because of this? Or do you try to influence their direction positively? I think both China and Vietnam are countries moving in the right direction. They have communist market economies. That has not translated to all the people yet, but I think I can make a strong case that things are better in China and Vietnam today than they were 20 years ago. That is the way we can change things. We cannot change things in the manner George Bush indicated when he said, "we are going to put a democracy in Iraq and everyone will be happy, and it's going to flower and there will be democracies everywhere." The world is not built that way. If there are flagrant abuses of human rights, then you know about it and you have to deal with it. But it is always a tough call for any American company. But I think that American companies, of all national companies, are far more tuned into being responsive to these big issues. We are one of the few countries with a law prohibiting bribing foreign officials--the Foreign Corrupt Practices Act. We are imperfect, but you have to weigh all these different factors. Many people want to talk in simple terms, "right" or "wrong." They don't understand; they are dangerous people. That is how you get the country in trouble.
GJIA: Our blog has run a number of articles on the South China Sea, which is being heralded as the next big hot spot for international concern. As a final question: Given the increasing naval ambitions of China in the region, our relations with Taiwan, and the economic motivators underlying the tensions, how should the United States proceed? How should it structure its future interactions with China over economic and geopolitical areas of contention?
Senator Hagel: We must continue to have a very strong navy. Seaways are critical. The South China Sea and the entire Indian Ocean through the Straits of Malacca are critical. America must maintain its pacific powerbase for the good of everyone. The countries of Southeast Asia are afraid of China. We must maintain commercial interests, maintain strong military relationships, and work with our allies. There is a strong future for us there. Obama has essentially carried on as Bush and Clinton did with the post-World War II focus on the Pacific that every administration has had. I think that that is the way we must continue to proceed. Obviously much of our economic future is tied to Asia, and we have huge interests there. We need to stay engaged in every way.
The Interview was conducted by Sikander Kiani, Michael Brannagan, and William Handel.
Chuck Hagel is a Distinguished Professor at Georgetown University and the University of Nebraska at Omaha. He served two terms in the United States Senate (1997-2009) representing the state of Nebraska. Senator Hagel was a senior member of the Senate Foreign Relations; Banking, Housing and Urban Affairs; and Intelligence Committees. He chaired the Foreign Relations International Economic Policy, Export and Trade Promotion Subcommittee; and the Banking Committee's International Trade and Finance, and Securities Subcommittees. He serves on the Boards of Directors of Chevron Corporation and Zurich's Holding Company of America; the Advisory Boards of Gallup, Deutsche Bank America and Corsair Capital. He also serves as Co-Chairman of the President's Intelligence Advisory Board, a member of the Secretary of Defense's Policy Board, and Secretary of Energy's Blue Ribbon Commission on America's Nuclear Future.


text:
Anyway, we have finish Ancient Rome and our now about to move onto the Middle Ages. The spine is Story Of The World so I have bought the activity book to try and add in some activities other than reading this term!
Here is a glimpse of a few of the things I have remembered to take photos of this term!
We made a model Roman ship and village to finish up our studies on Ancient Rome.
We went to the Annual Model Train and Lego Exhibit. Liam, who is still lego mad, was in his element. He could have stayed for hours just staring at the all the Lego exhibits. The others enjoyed it but not nearly as much as Liam!
Liana has started going to a sewing class at a friend's house every week. She is really enjoying it and has made a pleated blouse and skirt so far.
We also had our Annual Homeschool Ceilidh Dance and concert. Here is a picture of Liana and one of her friends in their costumes ready to perform.
And to finish, some random pics of my two big kids. Just because!


text:
Hoppy Smalls
Style: Small Beer
Availability: Special Release
We teamed up with Four Star Farms in Northfield, MA to produce this single-estate beer that highlights the incredible taste of their hops. Hoppy Smalls is an aggressively hopped small beer featuring malt and Magnum, Sterling, Cascade, and Teamaker hops all grown less the 20 miles away from our brewery. This beer packs some really unique floral hop flavors into a tiny package, and we're already looking forward to next year's harvest. Come on in and pick up a growler before this limited release beer hops away until Spring!


text:
Spreading Solar in the Philippines
Our 90kW solar installation for Perry's Farm in Majayjay, Philippines.
As the price of solar continues its steady decline around the world and technological improvements drive greater system efficiency, the Philippines is ready to cut its ties to fossil fuels in favor of renewables.
While solar systems used to be more common in higher income residential villages, they are now accessible to a larger portion of the population. With a quick return on investment, residents can get their electricity free of charge for a majority of the system's lifetime. It is estimated that the Philippines could save around P100 billion annually by transitioning from the use of gas, oil, diesel, and coal to renewables.
Earlier this June, the Climate Change Commission began work alongside key government agencies to develop a plan for climate change mitigation and adaptation, and sustainable development. This plan includes how the country will reshape its energy policies to phase out coal and lay the ground for renewables.
"Our worthy task will allow us to fulfill our responsibilities, as public servants, to serve and protect our people from this global threat, and will inspire us, as human beings, with greater capability to sustain humanity," said vice chair of the CCC, Secretary Emmanuel de Guzman.
Several businesses, banks, environmentalists, and civic groups also recently gathered in Makati to attend a forum, "The Truth about Solar: Now Cheaper than Coal." At the close of this event, the participants signed a declaration of independence from fossil fuels in support of going 100-percent renewable by 2030. With incoming President Dutertecalling for a greater investment in renewables, the Philippine government is on the same page.
UGE is currently working on several projects in the Philippines to help this transition, providing solutions to critical energy issues such as rising energy prices and grid shortages. Our 90kW solar system gave Perry's farm in Majayjay a reliable source of energy with 20% savings. Such projects demonstrate the full spectrum of benefits a movement away from fossil fuels can offer.
The future of renewables in the Philippines is full of opportunity - we're proud to be an impactful part of it.


text:
Requirements for Skills that Make Any Products, Content, or Services Available for Purchase
This page outlines requirements for any skill that allows users to make a purchase on Alexa. The list is not exhaustive, and the guidelines may change over time.
- 1. Skills that Allow Users to Purchase Physical Products or Services
- 2. Skills that Allow Users to Purchase Digital Products, Content, or Services
1. Skills that Allow Users to Purchase Physical Products or Services
If your skill allows users to pay for non-digital products or services (e.g., retail products, food delivery, or tickets for travel, events, or movie tickets, even if the tickets are delivered digitally), it must meet the following minimum requirements. This list is not exhaustive and the guidelines may change over time.
a. The skill must use Amazon Pay or account linking and must note in the skill description which one the skill uses. If the skill uses account linking, it must pass the tests described in Section 2.3 Skills with Account Linking.
b. The skill's invocation name must match the seller name, and the skill must guide customers to use the seller name when invoking the skill, e.g., "Alexa, ask [seller name]…" or "Alexa, open [seller name]."
c. The skill must confirm the seller of record, products, delivery details, and order total, including any taxes or tip/delivery charges, via voice before completing the order.
d. If the skill uses account linking, after completing the order, the skill must confirm the transaction is complete via voice and must provide order details via email and a card in the in the Alexa companion app (see Section 3.3 Home Cards).
e. If the skill uses account linking and sells an item(s) that costs $100 or more (or has an average transaction amount of $100 or more), it must give customers the option to set a four-digit voice code to protect against unwanted or accidental purchases. If customers have enabled this function, the skill must ask customers to confirm the code via voice before completing a transaction. Any skill that provides a voice code option should notify customers of the option to set up the code in their skill description.
To ensure the best experience for customers, we encourage all skills that allow users to pay for physical goods or services to participate in a beta test. This provides the developer an opportunity to incorporate customer feedback. See Skills Beta Testing for Alexa Skills for guidance on how to participate in a beta test.
2. Skills that Allow Users to Purchase Digital Products, Content, or Services
If your skill allows users to purchase digital products or services (such as premium features or content), it must meet the following minimum requirements. This list is not exhaustive and the guidelines may change over time.
a. The skill must use the Amazon In-Skill Purchasing feature.
b. The skill must offer free content that enables customers to assess the quality of the skill and understand the product for sale prior to purchasing. For child-directed skills that offer a subscription, the free content must be provided outside of a free trial.
c. Entitlements purchased by a customer must be usable by that customer for as long as your Alexa skill is made available. Consumables must be usable until fully consumed. Subscriptions must be supported through the final subscription term purchased by customers.
d. The skill description and example phrases cannot tell the customer how to purchase, or encourage them to purchase, the premium content. The skill description can only describe what is included in the premium content.
e. The list price for all in-skill purchases cannot exceed, at any time, the lowest list price or actual price you set for that content or functionality on any similar service.
f. Skills that enable customers to play casino-style games cannot offer consumable in-skill products within those games.
g. The In-Skill Purchasing feature cannot be used to solicit donations from end users. If you're a charity and you would like to receive donations through Alexa, visit Alexa Donations with Amazon Pay.
h. Skills offering in-skill purchasing cannot duplicate another published skill.
i. The skill cannot include upsell messages for in-skill products in notifications or reminders.
j. If the skill or the in-skill product(s) only work on a device with a screen, the skill must include the following disclaimer: "This [insert premium content description here] can only be accessed on a device with a screen."
- Entitlements and consumables: include the disclaimer in the purchase prompt description
- Subscriptions: include the disclaimer in the upsell message
k. At this time, in-skill purchasing is available only for Alexa skills in the US, the UK, Germany, and Japan
. Developers based in India are unable to add in-skill purchasing to their skills in any locale at this time.
l. At this time, in-skill purchasing in child-directed skills is available only in the US. Child-directed skills cannot include direct exhortations to children to buy items or persuade their parents to buy items for them and must not exploit children's inexperience, vulnerability, or naivety. Child-directed skills also must not include any pay-to-win mechanics that require a player to pay in order to either advance or avoid a negative consequence (for example, if a skill involves caring for a puppy, telling the user that if they don't buy the puppy a food in-skill product, the puppy will get sick). Child-directed skills cannot offer consumable in-skill products within those skills.


text:
There's so much to remember when you're planning a funeral, and it can be such an emotional time, it can be easy to forget things.
We've created this online checklist to be a quick reference list of all of the items involved in funeral planning.
You can print this list out and keep a copy to fill in, or you can complete the form online and save your progress.
To save the online version, you just need to register a username and password, and then enter these when you return to the website.
Our website works on computers, tablets and mobile phones, so wherever you are (with an internet connection) you can access this list.
We hope you find it helpful!


text: Click Here to download ERS-2014-05-14 #366 For those of you attending the CECA Conference in Quebec, have a great time! Wish I could be there but there's just too much going on to have been able to get away. I hope you all learn a lot, enjoy the vendor show and leave with more French in you than you came with. As a reminder, the email newsletter is going out however it is being pushed through our website. To subscribe, please visit www.elevatorradioshow.com and enter your email address in the box, upper right side, then click on the subscribe button and you'll be all set. For those of you who are visual learners like myself, click on the photo to the right. New/Article Links: Elevator shuts down 24 hours within being turned over Sad, Elevator video shows confrontation Crammed lifts are Passe Stone India Ltd, enters lift market Swallowed by a mechanical monster? I think not Boy trapped by escalator Elevator World announces acquisition EW Unplugged Photo of the week Well at least it's a comedy 157 Killed in mine accident 10 year old helped from stalled elevator Watching an elevator slip traction Adjusting a relay Viral Video People leave our equipment alone


text:
What We Are Reading
Alexandra Carmichael
October 12, 2012
This collection of links is inspired by NPR's #bestthingallweek hashtag on Twitter. What's the best thing you read this week?
Behavior Change is Belief Change by Buster Benson: "Anyone that tells you that you just need to 'walk one more bus stop every morning' in order to be a healthier person has reversed the formula (putting the easy thing first) in order to sell it to you. In order to be a person who walks to the bus stop every morning you have to change your core identity of yourself into a 'healthier person who walks a lot.'"
Startup = Growth by Paul Graham: "Usually successful startups happen because the founders are sufficiently different from other people that ideas few others can see seem obvious to them… We usually advise startups to pick a growth rate they think they can hit, and then just try to hit it every week."
Why Self-Discipline Is Overrated: The (Troubling) Theory and Practice of Control from Within by Alfie Kohn: "Just because motivation is internal doesn't mean it's ideal. If [people] feel controlled, even from within, they're likely to be conflicted, unhappy, and perhaps less likely to succeed (at least by meaningful criteria) at whatever they're doing."
Quantifying Kids, an interview with Bill Schuller: "A child's performance in the educational system is quantified at every step of the way. The difference in what I am doing is increasing the fidelity, increasing the time horizon, drawing correlation between the data and teaching my children to look at the results and reflect on them."
Building that Perfect Quantified Self App: Notes to Developers, Part 1 by Measured Me: "The diet logs now are not just about counting calories, but also tracking allergies and more serious ailments, and effects of ingredients on mental and physical performance, or sleep. Happiness has been already tied to location, and will soon probably be linked to the weather, quality of sleep, or activities on Facebook."
Growth Hacking – Lean Marketing for Startups by Mattan Griffel: This is a great slide deck talking about "a set of tactics and best practices for dealing with the problem of user growth." Thanks to Chris Hogg for finding it!