The present invention relates to the halogenated xanthine derivatives and the preparing method thereof. In particular, the present invention relates to the halogenated xanthine derivatives for anti-cancer and anti-metastasis and the preparing method thereof, and the halogenated xanthine derivatives further can be labeled with isotopic halogens.
The growth and proliferation of the prostate carcinoma relate to the gene regulations of androgen and androgen receptor (AR). Cai et al. (2007) reported that the α1-subunit of solubale guanylyl cyclase (sCG), composed of an α-subunit and a β-subunit, is a new androgen regulation gene. Solubale guanylyl cyclase wildly regulates the cellular function of nitric oxide and plays an important role in signaling transduction in animals and plants (Krumenacker et al., 2004). Nitric oxide combines with and activates sGC resulting in guanosine 5′-triphosphate (GTP) converted as 3′,5′-cyclic guanosine monophosphate (cGMP). Next, cGMP activates a serious of proteins, including ion channels, protein kinases and phosphodiesterase (PDE) (Papandreou et al., 1998). Androgen can stimulate sGCα1 expression in the prostate carcinoma. If sGCα1 expression is ceased, androgen-dependent and androgen-independent AR-positive receptors will stimulate the growth of the prostate carcinoma, and the individual sGCα1 over-expression will stimulate the proliferation of prostate carcinoma.
The interaction of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and Rho-A-participated cell signaling play the important role in the human prostate carcinoma PC-3 (Chen et al., 2005). In the human prostate carcinoma PC-3, cAMP/PKA can stimulate the Rho-A activation, and serine188 phosphorylation of Rho-A is necessary to the Rho-A activation. Since Rho-A participates the rearrangement of actin cytoskeleton, including cell attachment and movement, the interaction of cAMP/PKA and Rho-A cell signaling will change the cellular morphologyand the changes of cytoskeleton, migration and anchorage-independent growth.
In addition, the regulation pathways of sGC/cGMP/protein kinase G (PKG) and Rho kinase (ROCK2) are important on prostate smooth muscle tension. The inventor of the present invention found that PDE5A/ROCK2 inhibitor, 7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (abbreviated as KMUP-1), can inhibit the growth of the human prostate epithelial carcinoma PZ-HPV-7 (Liu et al., 2007). KMUP-1 is a synthetic xanthine derivative. In the past researches, it has been proved that KMUP-1 can increase the amount of cyclic nucleotide, inhibit PDE, activate potassium ion channel resulting in relaxations in aortic (Wu et al., 2001), corporeal carvenosa (Lin et al., 2002) and tracheal smooth muscles (Wu et al., 2004). KMUP-1 inhibits the phenylephrine-induced contraction, has effective inhibition activity of α1A/α1D-adenoreceptor combination, increases the amount of cAMP/cGMP, and increases phenylephrine-induced ROCK2 expression. KMUP-1 can inhibit the cellular growth of PZ-HPV-7 cells resulting in the cease of cell cycle at G0/G1 phase and increase p21 expression. The inventor of the present invention proved the knowledge of sGC/cGMP/PKG and ROCK2 regulation on the relaxation and proliferation of prostate. However, the anti-tumor and anti-tumor metastatic mechanisms of the prostate epithelial carcinoma are unknown (Liu et al., 2007).
It is therefore attempted by the applicant to deal with the above situation encountered in the prior art.
In accordance with one aspect of the present invention, a halogenated xanthine derivative is provided in the present invention. The halogenated xanthine derivative has a structure as shown in the following formula I.
R1 represents a first substituted group selected from a group consisting of a first hydrogen group, a first halide group and a C1-C10 alkyl group, and each of R2, R3, R4, R5 and R6 represents a second substituted group being one of a second hydrogen group and a second halide group. Each of the first halide group and the second halide group has one halide atom selected from a group consisting of a chloride (Cl), a bromide (Br) and an iodine (I).
Preferably, R1, R2, R3, R4, R5 and R6 have an identical radioactive halide group.
Preferably, the identical radioactive halide group is one radioactive halide atom selected from a group consisting of a radioactive chloride, a radioactive bromide and a radioactive iodine.
Preferably, R1, R2, R3, R4, R5 and R6 are different radioactive halide groups.
Preferably, each of the different radioactive halide groups is one radioactive halide atom selected from a group consisting of a radioactive chloride, a radioactive bromide and a radioactive iodine.
Preferably, each of R1, R2, R3, R4, R5 and R6 is a radioactive halide group selected from a group consisting of a 38Cl, a 37Cl, a 75Br, a 76Br, a 77Br, a 82Br, a 122I, an 123I, an 124I, an 125I and an 131I.
Preferably, the halogenated xanthine derivative has a function selected from a group of inhibiting a cancer, inhibiting a cancer metastasis and a combination thereof.
In accordance with another aspect of the present invention, a method for preparing a halogenated xanthine derivative is provided. The halogenated xanthine derivative has a structure as shown in the following formula II.
the method include a step of (a) reacting a theophylline with a piperazine in a first solution to obtain the halogenated xanthine derivative. R1 represents a first substituted group being one of a first hydrogen group and a first halide group, R7 represents a second substituted group being one of a second hydrogen group and a second halide group, and R2 represents a third substituted group being one of a third hydrogen group and a third halide group.
Preferably, the method further includes a step of: (b) radio-labeling a radioactive halogen group from a radioactive halide compound on at least one of R1, R2 and R7 of the halogenated xanthine derivative to obtain a radioactive halogenated xanthine derivative.
Preferably, the radioactive halide compound is one of a radioactive halogen, a radioactive sodium halide and a combination thereof which are dissolved in a second solution.
Preferably, the second solution is an organic solvent selected from a group consisting of a tetrahydrofuran, a methanol and an ethanol.
Preferably, the first solution is an organic solvent selected from a group consisting of a tetrahydrofuran, a methanol and an ethanol.
In accordance with another aspect of the present invention, a precursor of a halogenated xanthine derivative is provided. The precursor has a structure as shown in the following formula III.
R8 represents a first substituted group selected from a group consisting of a first hydrogen group, a first C1-C12 alkyl group, a first C1-C12 alkenyl group and a first C1-C12 dihaloalkyl group, R9 represents a second substituted group selected from a group consisting of: a second hydrogen group, a second C1-C12 alkyl group, a second C1-C12 alkenyl group, a monohaloalkyl group and a second C1-C12 dihaloalkyl group, and X represents one of a halide group and a radioactive halide group.
Preferably, the halide group is a halide atom selected from a group consisting of a chloride (Cl), a bromide (Br) and an iodine (I), and the radioactive halide group is a radioactive halide atom selected from a group consisting of a radioactive chloride, a radioactive bromide and a radioactive iodine.
In accordance with another aspect of the present invention, a precursor of a halogenated xanthine derivative is provided. The precursor has a structure as shown in the following formula IV.
R1 represents a first substituted group being one of a hydrogen group and a halide group, and R10 represents a C1-C12 iodoalkyl group.
Preferably, the halide group is one halide atom selected from a group consisting of a chloride (Cl), a bromide (Br) and an iodine (I).
Preferably, the precursor is obtained by reacting a dihaloalkane with one of a theophylline and a chlorotheophylline.
Preferably, an iodide group of the C1-C12 iodoalkylgroup of the precursor is further radio-labeled a first radioactive halide group.
Preferably, the halide group is further radio-labeled a second radioactive halide group when R1 of the precursor is the halide group.
Preferably, the precursor is an iodomethyl chlorotheophylline when R1 is the chloride group and R10 is an iodomethyl group.
The halogenated xanthine derivative and the preparing method thereof in the present invention, especially relative to the halogenated xanthine derivative for anti-cancer and anti-metastasis and the preparing method thereof, further can be labeled the radioactive halogen thereon. The prepared radioactive pharmaceuticals not only use in clinical diagnosis and clinically radioactive reagent, but also use in labeled pharmaceuticals.
The effect of detection and quantity can be easily achieved using the radioactivity of radioactive isotope and radioactive isotope detector. For instance, radioactive iodine (131I, 125I) has been widely used in the detection of radioactive material in the clinical diagnosis and treatment. With the design and determination of computer software, medical images progress from the present two-dimensional image to three-dimensional image and the information observation such as image variation. The halogenated xanthine derivative in the present invention selects different halogenated radioactive element, such as 38Cl, 37Cl, 75Br, 76Br, 77Br, 82Br, 122I, 123I, 124I, 125I, and 131I, simultaneously on the substituted group of different position, so as to improve detection, diagnostic tracing and treatment of anti-cancer.
The above objectives and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed descriptions and accompanying drawings, in which:
The present invention will now be described more specifically with reference to the following Embodiments. It is to be noted that the following descriptions of preferred Embodiments of this invention are presented herein for purpose of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.
Twenty grams of 7-ethylchloro-theophylline is added into a flask containing 50 ml of tetrahydrofuran (THF) and 60 ml of 1-(2-chlorophenyl)-piperazine, and a suitable amount of sodium carbonate (Na2CO3) is added therein. The flask is connected with a condensing tube, and the reaction mixture in the flask is heated to boil to reflux for 2 hours. The reaction mixture is cooled after the reaction is completed, the product is dissolved and eluted with ethanol, and the elutant is collected, concentrated and heated under the water bath. The elutant is filtered while heated, and the filtered solution is cooled and the white crystal product is obtained. The white crystal product is 7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (abbreviated as KMUP-1). The reaction formula is illustrated as follows. In addition, tetrahydrofuran can be substituted for methanol or ethanol.
Twenty grams of 2-ethylbromo-theophylline is added into a flask containing 50 ml of tetrahydrofuran and 60 ml of 1-phenylpiperazine, and a suitable amount of sodium carbonate is added therein. The flask is connected with a condensing tube, and the reaction mixture in the flask is heated to boil to reflux for 2 hours. The reaction mixture is cooled after the reaction is completed, and the product is dissolved and eluted with ethanol. The elutant is collected, concentrated and heated under the water bath. The elutant is filtered while heating, and the filtered solution is cooled and the white crystal product is obtained. The white crystal product is 7-[2-[4-(2-phenyl)-piperazinyl]ethyl]-1,3-dimethylxanthine (abbreviated as KMUP-H), and the chemical formula is illustrated as follows.
Twenty grams of 7-ethylbromo-8-chloro-theophylline is added into a flask containing 50 ml of tetrahydrofuran and 60 ml of 1-(2-chlorophenyl)piperazine, and a suitable amount of sodium carbonate is added therein. The flask is connected with a condensing tube, and the reaction mixture in the flask is heated to boil to reflux for 2 hours. The reaction mixture is cooled after the reaction is completed, and the product is dissolved and eluted with ethanol. The elutant is collected, concentrated and heated under the water bath. The elutant is filtered while heating, and the filtered solution is cooled and the white crystal product is obtained. The white crystal product is 7-[2-[4-(2-chlorophenyl)-piperazinyl]ethyl]-1,3-dimethyl-8-chloroxanthine (abbreviated as KMUP-2Cl), which has a chemical formula as follows.
1. Twenty grams of 2-ethylbromo-theophylline is added into a flask containing 50 ml of tetrahydrofuran and 60 ml of 1-(2-iodophenyl)piperazine, and a suitable amount of sodium carbonate is added therein. The flask is connected with a condensing tube, and the reaction mixture in the flask is heated to boil to reflux for 2 hours. The reaction mixture is cooled after the reaction is completed, and the product is dissolved and eluted with ethanol. The elutant is collected, concentrated and heated under the water bath. The elutant is filtered while heating, and the filtered solution is cooled and the white crystal product is obtained. The white crystal product is KMUP-I, which has a structural formula as follows.
2. Twenty grams of KMUP-H is added into a flask containing 50 ml of tetrahydrofuran and 60 mg of iodine (I2), and a suitable amount of sodium carbonate is added therein. The flask is connected with a condensing tube, and the reaction mixture in the flask is heated to boil to reflux for 2 hours. The reaction mixture is cooled after the reaction is completed, and the product is dissolved and eluted with ethanol. The elutant is collected, concentrated and heated under the water bath. The elutant is filtered while heating, and the filtered solution is cooled and the white crystal product is obtained. Iodine can be substituted as the mixture of potassium iodide (KI) and I2 dissolved in THF. The reaction formula of KMUP-I is illustrated as follows.
3. The equal amount of non-radioactive KMUP-I and radioactive sodium iodide (NaI*) are mixed in tetrahydrofuran to exchange the radioactivity with each other, so as to obtain the isotope-labeled KMUP-I (KMUP-I*).
1. Twenty grams of 2-propylbromo-theophylline is added into a flask containing 50 ml of tetrahydrofuran and 60 ml of 1-(3-chlorophenyl)piperazine, and a suitable amount of sodium carbonate is added therein. The flask is connected with a condensing tube, and the reaction mixture in the flask is heated to boil to reflux for 2 hours. The reaction mixture is cooled after the reaction is completed, and the product is dissolved and eluted with ethanol. The elutant is collected, concentrated and heated under the water bath. The elutant is filtered while heating, the filtered solution is cooled, and the white crystal product is obtained.
2. Twenty grams of theophylline is added into a flask containing 50 ml of tetrahydrofuran and 60 ml of 1-(3-chlorophenyl)-4-(3-chloroproyl)-piperazine HCl, and a suitable amount of sodium carbonate is added therein. The flask is connected with a condensing tube, and the reaction mixture in the flask is heated to boil to reflux for 2 hours. The reaction mixture is cooled after the reaction is completed, and the product is dissolved and eluted with ethanol. The elutant is collected, concentrated and heated under the water bath. The elutant is filtered while heating, the filtered solution is cooled, and the white crystal product is obtained.
Twenty grams of KMUP-2Cl is added into a flask containing 50 ml of tetrahydrofuran and 60 ml of I2, and a suitable amount of sodium carbonate is added therein. The flask is connected with a condensing tube, and the reaction mixture in the flask is heated to boil to reflux for 2 hours. The reaction mixture is cooled after the reaction is completed, and the product is dissolved and eluted with ethanol. The elutant is collected, concentrated and heated under the water bath. The elutant is filtered while heating, the filtered solution is cooled, and the yellow crystal product is obtained. Iodine (I2) can be substituted for the mixture of potassium iodide (KI) and I2 dissolved in tetrahydrofuran.
1. Twenty grams of KMUP-2Cl is added into a flask containing 50 ml of tetrahydrofuran and 60 ml of radioactive I2, and a suitable amount of sodium carbonate is added therein. The flask is connected with a condensing tube, and the reaction mixture in the flask is heated to boil to reflux for 2 hours. The reaction mixture is cooled after the reaction is completed, and the product is dissolved and eluted with ethanol. The elutant is collected, concentrated and heated under the water bath. The elutant is filtered while heating, the filtered solution is cooled, and the yellow crystal product is obtained. Iodide (I2) can be substituted for the mixture of potassium iodide (KI) and I2 dissolved in tetrahydrofuran.
2. The equal amount of non-radioactive KMUP-I2Cl and radioactive sodium iodide (NaI) are mixed in tetrahydrofuran to exchange the radioactivity with each other, so as to obtain the radio-labeled KMUP-I2Cl (KMUP-I2Cl*).
The precursor of the halogenated xanthine derivative for anti-cancer is provided in the present invention, and the precursor thereof has a structural formula as follows, in which R8 is a substitutive group selected from a group consisting of a hydrogen group (H), an alkyl group (CnH2n+1), an alkenyl group and a dihaloalkyl group. The number of carbon atom of the alkyl group, the alkyenyl group and the dihaloalkyl group is ranged between 1 and 12 (1≦n≦12), R9 is one of a hydrogen group, an alkyl group, an alkenyl group, a monohaloalkyl group and a dihaloalkyl group. The number of carbon atom of the hydrogen group, the alkyl group, the alkenyl group, the monohaloalkyl group and the dihaloalkyl group is ranged between 1 and 12 (1≦n≦12). X is one substitutive group selected from a group consisting of a halide group (X) and a radioactive halide group (X*). The halide group includes one of a chloride and an iodide, and the radioactive halide group includes a radioactive chloride and an radioactive iodide.
The precursors of the halogenated xanthine derivatives in the present invention includes at least two chemicals nominated as [1-alkyl,4-(2-halophenyl)]piperazine and [1(2-halophenyl),4-methyl]piperazine, such as 2-iodophenyl piperazine and [1(2-iodophenyl),4-methyl]piperazine.
The precursor of the halogenated xanthine derivative for anti-cancer is further provided in the present invention, and the precursor has a structural formula as follows, in which R1 is one of a hydrogen group (H) and a chloride group (Cl), and R10 is one substitutive group selected from a group consisting of an iodoalkyl group (CnH2nI) which the number of carbon atom is ranged between 1 and 12 (1≦n≦12). The precursor is obtained by reacting dihaloalkane with theophylline or chlorotheophylline.
The chemical formula of one of the precursor in this embodiment is iodomethyl chlorotheophylline (iodocaffeine), which has a structural structure as follows.
The precursor in this embodiment can be radio-labeled using radioactive halogen, such as the radio-labeled iodomethyl chlorotheophylline, which is shown as the following structural formula.
Cells at a density of 105 cells/well are seeded in 24-well cell culture plate to 90% confluence, and KMUP-2Cl or KMUP-1 at different concentrations (0.1, 1, 10 and 100 μM) is added therein. The cells are incubated at 37° C. and an atmosphere of 5% CO2 for 24 and 48 hours, then the medium is replaced with 490 μl of fresh medium and 10 μl of 5 mg/ml of methylthiazolyldiphenyl-tetrazolium bromide (MTT). After the reaction mixture is mixed well, the cell culture plate is covered with the aluminum foil and further is incubated in the incubator for 2.5 hours. The MTT solution is withdrawn while the reaction time is finished, 500 μl of acidified isopropanol is added into the well to dissolve the crystal violet, formazan. Two hundred microliter of the supernatant is transferred to another new 96-well cell culture 10 minutes later to determine the absorbance at the wavelengths of 540 nm (OD540) and 630 nm (OD630) using spectrophotometer (Hitachi U-200, Japan). The drug-treated cell survival rate is evaluated by comparing the value of OD540-OD630 in each group with that in the control group. The cell survival rate represents the toxicity of the drug to the cells.
Tongue squamous cell carcinoma SCC-25 (105 cells/well) is seeded in 6-well cell culture plate to 90% confluence, then KMUP-2Cl at different concentrations (0.1, 1, 10 and 100 μM) respectively is added therein. The cells are incubated at 37° C. and an atmosphere of 5% CO2 for 24 and 48 hours. The cell survival rate is determined by trypan blue exclusion assay. Firstly, 20 μl cell supernatant is mixed with 20 μl of trypan blue, then 15 μl mixture is added into the groove of the hemocytometer which has a cover lid thereon. The cells are counted under the microscope, and the live cells are not stained but the dead ones are blue. The total cells in four squares are counted, then the numbers is divided by four, multiplied with the dilution rate (at least 2 because of mixing with the equal volume of trypan blue). Finally, the number is multiplied with 104, and the result is the total cell numbers in the cell supernatant per milliliter.
The counted cells (105 cells/ml×3 ml) are seeded in the 6-well cell culture plate for 24 hours for attaching on the bottom of the cell culture plate, then DMEM/F12 medium without fetal bovine serum (FBS) is added therein for further incubation for 24 hours. Next, the medium is replaced with DMEM/F12 supplemented with 10% FBS, and the drugs at different concentrations are respectively added therein for reacting 24 and 48 hours. The cellular inoculate is harvested, and the wells are washed with phosphate buffered saline (PBS) for twice or triplet. The cells are eluted with 0.25% trypsin-0.02% EDTA, and are collected with the original inoculate in the centrifuge tube which is centrifuged at 4° C. at 1250×g (1200 rpm) for 5 minutes. Next, the supernatant is discarded and the pellet is resuspended in 1 ml PBS and the resuspended cells are transferred to an eppendorf tube which is centrifuged once again. The supernatant is discarded, and the cells are fixed with 300 μl PBS (4° C.) and 700 μl of 99.5% ethanol (50, 50, 100, 100, 200 and 200 μl ethanol respectively and sequentially added to make the cells resuspended in the well) for 30 minutes. The supernatant is discarded after centrifuging, then 570 μl PBS, 2 μl of 10 μg/ml ribonuclease (RNase) and 30 μl of 0.5% Triton are added therein and reacted at 37° C. for 1 hour. The supernatant is discarded after centrifuging, then 600 μl PBS and 1 μl of 10 μg/ml propidium iodide (PI) are added therein. The tube is rotated at 4° C. for 15 to 30 minutes on the DS LAB Rotator. The cells are transferred into a tube, and the cells are detected using CyFlow® cytometer. The ratios of G0/G1, S and G2/M phases are determined using multicycle DNA analysis software.
The amount of protein is determined by Bio-Rad protein reagent. Bovine serum albumin (BSA) of 0.1 mg/ml is the protein standard in the protein assay. Distilled water of 240 μl containing various known protein standards (the amounts of protein respectively are 0, 2, 4, 8, 12, 16, 20 and 30 μg) and 60 μl of Bio-Rad reagent are mixed gently in 96-well cell culture plate. The absorbance at the wavelength of 595 nm relative to the absorbance of blank test is determined using spectrophotometer (Hitachi U-200, Japan). A standard curve is evaluated from the known amounts of the protein standards and the absorbance thereof, and the amounts of the unknown proteins can be obtained according to this standard curve.
The quantified proteins are resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein expression is evaluated by immunoblotting. The dilution rate of primary antibodies (i.e. anti-AR, anti-p21, anti-p27, anti-cyclin D, anti-cdk4 and anti-cdk6) is 1000 times, and that of anti-β-actin is 5000 times. The primary antibody detects the proteins for 1 hour, and the primary antibody is detected with the horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour. Finally, the detected protein are developed with enhanced chemical luminescence.
1. All the abovementioned experiments are at least repeated independently for triplet, and the results are shown as mean±s.e.m. The data are analyzed by ANOVA (analysis of variance), then are analyzed with Dunnett's test. P-value is a comparison comparing with the vehicle, and p-value<0.05 means the statistic significance and is shown as a star symbol (*). The vehicle is a solution containing propylene glycol, 0.2% ethanol, 0.58% dimethyl sulfoxide (DMSO) and 0.02% hydrogen chloride.
2. The calculations of 50% effective dosage (ED50) and 95% confidential interval use Litchfield and Wilcoxon method.
The cell lines utilized in the present invention are listed in Table 1.
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p27 can combine with Cyclin D, or combine with the catalytic subunit CDK4 of Cyclin D to form a complex. p27 can inhibit the catalytic activity of CDK4. The increased amount of p27 would induce the cell cycle to stop in G1 phase. Identically, p27 also can combine with other CDK proteins while p27 and other Cyclin subunit (such as Cyclin E/CDK2 and Cyclin A/CDK1) forms the complex.
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In addition to observe the inhibition effect of the halogenated xanthine derivatives on the cancer cell lines in the cellular level, the animal experiments are further performed. LNCaP-xenografted rude mice are injected intraperitoneally with KMUP-1 (150 and 150 mg/kg) and dosed orally with KMUP-1 (200 and 400 mg/kg) respectively once per day, and a continuity for two months. Comparing with the control, the intraperitoneal injection of KMUP-1 can significantly eliminate the LNCaP prostate tumor in the xenografted rude mice. The significant effect can be obtained (data not shown) when KMUP-1 is 100 mg/kg. The oral dosage of KMUP-1 also can significantly eliminate the LNCaP prostate tumor in the xenografted rude mice. The significant effect can be obtained (data not shown) when KMUP-1 is 200 mg/kg.
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Accordingly, the xanthine derivatives and the halogenated xanthine derivatives prepared in the present invention can effectively inhibit the growth and the metastasis of carcinoma in vitro, and can effectively inhibit the growth of prostate tumor and even decrease the tumor size in vivo. In particular, the xanthine derivatives and the halogenated xanthine derivatives of the present invention have significant inhibition effect on cell lines from different human tissues.
While the invention has been described in terms of what is presently considered to be the most practical and preferred Embodiments, it is to be understood that the invention needs not be limited to the disclosed Embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims, which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.