Heat the mixture gently until the reaction commences, and remove the flame. Shake the flask frequently and take care that the insoluble acid adhering to the sides of the flask is transferred to the reaction mixture: occasional gentle warming may be necessary. After about 20 min, most of the tin will have reacted and a clear solution remains.
Allow to cool somewhat and decant the liquid into a 1-litre beaker; wash the residual tin by decantation with 15 ml of water, and add the washings to the contents of the beaker.
Add concentrated ammonia solution d 0. Transfer the filter cake to a beaker, heat on a water bath with ml of water to ensure extraction of the product and refilter. Concentrate the combined filtrate and washings until the volume has been reduced to ml: filter off any solid which separates.
Furthermore, dependence of the release mechanism of the drug upon biodegradation may cause interpatient variability. Alternatively, the drugs may be conjugated to a carrier through permanent covalent bonds.
This approach is applied to various classes of molecules, from so-called small molecules, through natural products up to larger proteins. Liraglutide is an example of a peptide drug that achieves an extended half-life by permanent covalent modification with a palmitoyl moiety. The fatty acid alkyl chain serves to provide albumin binding in vivo and the palmitoylated peptide forms an albumin complex that acts as a drug reservoir in the blood stream.
Albuferon is an example of a protein drug that achieves an extended half-life by permanent covalent modification with another protein that in itself has a long half-life. The corresponding fusion protein of albumin and interferon alpha, Albuferon, exhibits a significantly extended half-life as compared to interferon alpha.
Many small molecule medicinal agents, like alkaloids and anti-tumor agents, show low solubility in aqueous fluids. One way to solubilize these small molecule compounds is to conjugate the small molecule compounds to hydrophilic water-soluble polymers. A variety of water-soluble polymers, such as human serum albumin, dextran, lectins, poly ethylene glycol PEG , poly styrene-co-maleic anhydride , poly N- hydroxypropylmethacrylamide , poly divinyl ether-co-maleic anhydride , hyaluronic acid have been described for this purpose R.
Duncan, Nature Rev. Drug Disc, , 2, Covalent modification of biological molecules with poly ethylene glycol has been extensively studied since the late s. So-called PEGylated proteins have shown improved therapeutic efficacy by increasing solubility, reducing immunogenicity, and increasing circulation half-live in vivo due to reduced renal clearance and proteolysis by enzymes see, for example, Caliceti P.
Drug Deliv. However, many biological molecules such as IFN alfa 2, saquinavir or somatostatin are inactive or show decreased biological activity when a carrier is covalently conjugated to the drug T. Peleg-Shulman et al, J. In order to avoid shortcomings imposed by either the non-covalent polymer mixtures or the permanent covalent attachment, it may be preferable to employ a prodrug approach for chemical conjugation of the drug to the polymer carrier.
In such polymeric prodrugs, the biologically active moieties drugs, therapeutic, biological molecule, etc. Prodrugs are therapeutic agents that are almost inactive per se but are predictably transformed into active molecular entities see B. Testa, J. The carrier prodrug approach may be applied in such a fashion that the drug is released in vivo from the polymer in order to regain its biological activity.
The reduced biological activity of the prodrug as compared to the released drug is of advantage if a slow or controlled release of the drug is desired. In this case, a relatively large amount of prodrug may be administered without concomitant side effects and the risk of overdosing. Release of the drug occurs over time, thereby reducing the necessity of repeated and frequent administration of the drug.
Prodrug activation may occur by enzymatic or non-enzymatic cleavage of the temporary bond between the carrier and the drug molecule, or a sequential combination of both, i. In an enzyme-free in-vitro environment such as an aqueous buffer solution, a temporary bond such as an ester or amide may undergo hydrolysis, but the corresponding rate of hydrolysis may be much too slow and thus outside the therapeutically useful range. In an in vivo environment, esterases or amidases are typically present and the esterases and amidases may cause significant catalytic acceleration of the kinetics of hydrolysis from twofold up to several orders of magnitude see, for example, R.
Greenwald et al. Prodrugs fall in two classes, bioprecursors and carrier- linked prodrugs. Bioprecursors do not contain a carrier group and are activated by the metabolic creation of a functional group. In carrier-linked prodrugs the active substance is linked to a carrier moiety by a temporary linkage.
The carrier may be biologically inert for instance PEG or may have targeting properties for instance antibodies. This invention is concerned with polymeric carrier-linked or macromolecular prodrugs, where the carrier itself is a macromolecule such as a carrier protein or polysaccharide or poly ethylene glycol. Cleavage of a carrier prodrug generates a molecular entity drug of increased bioactivity and at least one side product, the carrier. After cleavage, the bioactive entity will reveal at least one previously conjugated and thereby protected functional group, and the presence of this group typically contributes to the drug's bioactivity.
In order to implement a prodrug strategy, at least one selected functional group in the drug molecule is employed for attachment of the carrier polymer. Preferred functional groups are hydroxyl or amino groups.
Consequently, both the attachment chemistry and hydrolysis conditions depend on the type of functional group employed. Numerous macro molecular prodrugs are described in the literature where the temporary linkage is a labile ester bond. In theses cases, the functional group provided by the bioactive entity is either a hydroxyl group or a carboxylic acid e.
Greenwald, A. Pendri, CD. Conover, H. Zhao, Y. Choe, A. Martinez, K. Shum, S. Guan, J. Especially for therapeutic biomacromolecules but also for certain small molecule drugs, it may be desirable to link the carrier to amino groups of the bioactive entity i. N-terminus or lysine amino groups of proteins. This will be the case if masking the drug's bioactivity requires conjugation of a certain amino group of the bioactive entity, for instance an amino group located in an active center or a region or epitope involved in receptor binding.
Also, during preparation of the prodrug, the amino groups may be more chemo selectively addressed and serve as a better handle for conjugating the carrier and the drug because of their greater nucleophilicity as compared to hydroxylic or phenolic groups.
This is particularly true for proteins and peptides which may contain a great variety of different reactive functionalities, where non-selective conjugation reactions lead to undesired product mixtures which require extensive characterization or purification and may decrease reaction yield and therapeutic efficiency of the product.
Amide bonds are usually much more stable against hydrolysis than ester bonds, and the rate of clevage of the amide bond would be too slow for therapeutic utility in a carrier- linked prodrug. Therefore it is advantageous to add structural chemical components such as neighbouring groups in order to exert control over the cleavability of the prodrug amide bond.
Such additional cleavage-controlling chemical structures that are provided neither by the carrier entity nor by the drug are termed "linkers". Prodrug linkers can have a strong effect on the rate of hydrolysis of a given temporary bond.
Variation of the chemical nature of these linkers allows the engineering of the properties of the linker to a great extent. Several examples have been published of the prodrug activation of amine-containing biologically active moieties by specific enzymes for targeted release. A prerequisite for enzymatic dependence is that the structure of the linker displays a structural motif that is recognized as a substrate by a corresponding endogenous enzyme.
In these cases, the cleavage of the temporary bond occurs in a one-step process which is catalyzed by the enzyme. Cavallaro et al. Bioconjugate Chem. Enzymatic release of cytarabin is effected by the protease plasmin which concentration is relatively high in various kinds of tumor mass.
Enzyme-catalyzed acceleration of prodrug cleavage is a desirable feature for organ or cellular targeting applications. Targeted release of the bioactive entity is effected, only if an enzyme, that selectively cleaves the linkage, is specifically present in the organ or cell- type chosen for treatment.
A major drawback of predominantly enzymatic cleavage is interpatient variability. Enzyme levels may differ significantly between individuals resulting in biological variation of prodrug activation by the enzymatic cleavage. The enyzme levels may also vary depending on the site of administration. For instance it is known that in the case of subcutaneous injection, certain areas of the body yield more predictable therapeutic effects than others.
To reduce this unpredictable effect, non-enzymatic cleavage or intramolecular catalysis is of particular interest see, for example, B.
Furthermore, it is difficult to establish an in vivo-in vitro correlation of the pharmacokinetic properties for enzyme-dependent carrier-linked prodrugs. In the absence of a reliable in vivo-in vitro correlation optimization of a release profile becomes a cumbersome task.
Other carrier prodrugs employing temporary linkages to amino groups present in the drug molecule are based on a cascade mechanism. Cascade cleavage is enabled by linker compounds that are composed of a structural combination of a masking group and an activating group.
The masking group is attached to the activating group by means of a first temporary linkage such as an ester or a carbamate. The activating group is attached to an amino-group of the drug molecule through a second temporary linkage, for instance a carbamate. The stability or susceptibility to hydrolysis of the second temporary linkage e. In the presence of the masking group, the second temporary linkage is highly stable and unlikely to release the drug with therapeutically useful kinetics.
In the absence of the masking group, this linkage becomes highly labile, causing rapid cleavage and drug release.
The cleavage of the first temporary linkage is the rate-limiting step in the cascade mechanism. This first step may induce a molecular rearrangement of the activating group such as a 1,6-elimination. The rearrangement renders the second temporary linkage so much more labile that its cleavage is induced.
Ideally, the cleavage rate of the first temporary linkage is identical to the desired release rate for the drug molecule in a given therapeutic scenario.
Furthermore, it is desirable that the cleavage of the second temporary linkage is substantially instantaneous after its lability has been induced by cleavage of the first temporary bond. Examples of polymeric prodrugs based on 1,6-elimination have been described by R.
DeGroot et al. Refluxing method to the reaction mixture will be used in the experiment in order to proceed the esterification reaction without decreasing the amount of solvent. To precipitate the product from solution, the PH of reaction mixture is made to eight. The product will be isolated by vacuum filtration and washed by distilled water. After it cooled to room temperature, 0. The reaction mixture was then refluxed for about 60 to 75 minutes at Upon the completion of reaction, saturated sodium bicarbonate 1M was added dropwise until the pH of solution was around 8.
The PH of solution was monitored by the PH paper. During the process of adding sodium bicarbonate, white precipitate of benzocaine formed. The reaction mixture was then vacuum filtrated to give benzocaine as white crystals 71 mg, IR ATR cm-1 : Discussion: The Fischer Esterification reaction proceeded without any notable chemical issues.
The precipitation of product from the solution and the vacuum filtration were also successful. The percentage yield of the reaction is The yield cannot be considered poor because the Fischer Esterification is a reversible reaction under acidic condition. Its impossible to run this reaction completely Although the refluxing process lasted about an hour, the refluxing process only extends the reaction time without proceeding the reaction. Different from the technique of distillation, the fractions of mixture are not removed in the refluxing process.
Because the product benzocaine is not removed from the mixture, the concentration of product increases during the whole process of the reaction. The decreasing concentration of starting material paminobenzoic acid and increasing concentration of product benzocaine impedes the reaction from moving to the forward direction.
The longer the reaction takes place, less and less products will form. The stretch at A lot of starting material remain with the products. But it does not show up in the IR spectrum.
No O-H stretch around means no carboxylic acid exists in the sample. The reason lies in the difference of solubility between two compounds in the basic environment. The starting material, p-aminobenzoic acid, is very soluble in the basic solution when it is deprotonated by the base and forms the water-soluble carboxylate anion. On the other hand, the benzocaine lacks the acidic protons and cannot be deprotonated by the base.
Since final pH of the solution was made to 8, all the starting material exist as an anion rather than the solid.Thus, the prodrugs which contain acidic syntheses can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium. But on the other hand I had to give the habitants the possibility to earn a minimum income, be interested in solo travel, and greater independence and. The invention of the present application is described in dxn business plan pakistan detail in the following sections.
The PH of solution was monitored by the PH paper. Rummel, A. Preferred functional groups are hydroxyl or amino groups.