Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-11-15
2002-07-02
Wilson, James O. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S027100, C536S027600, C536S027810, C536S028500, C536S028530, C536S028540
Reexamination Certificate
active
06414137
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the purification of nucleosides, and more particularly to a selective solvent extraction method for purifying protected nucleosides.
BACKGROUND OF THE INVENTION
Nucleosides are compounds of importance in physiological and medical research, obtained during partial decomposition, i.e., hydrolysis, of nucleic acids, and containing a purine or pyrimidine base linked to either D-ribose (forming ribonucleosides) or D-deoxyribose (forming deoxyribonucleosides). They are nucleotides minus the phosphate group. Well-known nucleosides include adenosine, cytidine, guanosine and thymidine. Nucleosides are multi-functional compounds, having both amino and alcohol functional groups. In order to conduct syntheses selectively and efficiently, it is necessary to block specific functional groups in order to achieve reaction at the desired sites. The “protecting” groups are designed to be removed under specific carefully controlled conditions, usually under relatively mild and typically acidic conditions. To be useful as precursors in the synthesis of high value pharmaceuticals, it is necessary that protected nucleosides be of very high purity (i.e., greater than about 99% by weight (wt %), preferably greater than about 99.5 wt %). The very sensitive nature of the protecting groups together with the variety of polar and non-polar impurities generated during the syntheses of these derivatives makes their purifications complicated, expensive, and difficult to scale-up to industrial scale production.
Typically, the protection of nucleosides involves the derivatization of both amino and alcohol functional groups. An exception is thymidine, which requires only the protection of alcohol groups. Various schemes are employed to achieve these protected nucleosides, but usually the N-protected derivatives (most often N-acylated) are isolated and purified before protecting the alcohol groups. The presence of the free alcohol groups often leaves these derivatives with sufficient polarity that they can be readily purified by recrystallization. However, when these alcohol groups are also derivatized (most often as trityl ethers), the fully protected nucleosides are usually very difficult to crystallize. Purification then is typically achieved by resorting to column chromatography, usually followed by precipitation of the appropriate column fractions into non-solvents to remove traces of co-eluted colored by-products.
Discussions of the synthesis and protection of nucleosides by derivatization may be found in many references, including the following, all of which are incorporated herein by reference. One method of protecting nucleosides is described in Ti, et al., “Transient Protection: Efficient One-flask Syntheses of Protected Deoxynucleosides,”
J. Am. Chem. Soc
., Vol. 104, 1316-1319 (1982), which is discussed in more detail below in regard to the examples. Other methods of synthesizing protected nucleosides are set forth in Charubala, et al., “Nucleotides XXIII: Synthesis of Protected 2′-Deoxyribonucleoside-3′-phosphotriesters Containing the p-Nitrophenylethyl Phosphate Blocking Group,” Synthesis 965,(1984). Still other methods for synthesizing such protected nucleosides are set forth in Kierzek, “The Synthesis of 5′-O-dimethoxytrityl-N-acyl-2′-deoxynucleosides, Improved ‘Transient 155- Protection’ Approach,” Nucleosides & Nucleotides, 4(5), 641-649 (1985). In all of these references, protection by N-acylation is effected with benzoyl chloride on adenosine and cytidine derivatives, and with isobutyric anhydride on guanosine derivatives, as is wellknown in the art. The compounds are then further protected by the introduction of methoxytrityl or dimethoxytrityl groups, also as is well-known in the art. An earlier article on the protection of such nucleosides may be found in Schaller, et al.,
J. Amer. Chem. Soc
., Vol. 85, 3821-3827 (1963). Another article on protected nucleosides is McGee, et al., “A Simple High Yield Synthesis of N
2
-(2-Methylpropanoyl)-2′-deoxyguanosine,” Synthesis, 540 (1983). In all of the reported syntheses, the protected nucleosides must be subjected to purification prior to their use in pharmaceutical syntheses.
The impurities generated during the various syntheses of protected nucleosides include polar compounds, such as isobutyric acid and benzamide, and non-polar compounds such as dimethoxytrityl methyl ether and the 3′,5′-bis-dimethoxytrityl ether nucleoside derivatives.
On a laboratory scale, recrystallization is widely practiced as a purification method. However, because of the broad range of polarity exhibited by these impurities, purification of protected nucleosides with a single recrystallization solvent system is difficult to achieve. Multiple recrystallizations are often required to achieve required purity levels. As an industrial process, losses (often greater than 10%) of valuable product to the recrystallization medium, and long processing times for mixing, heating, cooling and filtration make this method less attractive.
Column chromatography, especially flash silica gel chromatography, has been used extensively to purify protected nucleosides on a small scale. This method requires the use of large volumes of high purity solvents in proportion to the amount of material purified. The method is also labor-intensive, requiring precise monitoring to make the fraction cuts at the appropriate times to maximize yield of desired product. For these reasons, large-scale use of this method of purification can be very costly.
The equipment required to conduct flash silica gel chromatography on a multi-kilogram scale is expensive to purchase and operate. For example, one commercially available production scale chromatography unit is capable of separating up to about 4 kg of material per run. Run times can vary from 18 to 36 minutes, at an elution rate of 7 liters per minute. The basic unit investment is very expensive, coupled with the cost (and subsequent disposal cost) of 125 to 250 liters of expensive high purity solvent per run. These costs make purification by chromatography unattractive on an industrial scale.
DETAILED DESCRIPTION OF THE INVENTION
In the purification process of the present invention, solid particles of protected nucleosides are selectively washed to remove undesirable polar and/or non-polar impurities, while leaving the solid nucleoside particles substantially undissolved. In a preferred embodiment of the invention, the process comprises two slurry washing steps. In the first slurry washing step, the particles of protected nucleoside are slurried with a solvent for the polar impurities, in which solvent the particles are insoluble or, at most, only slightly soluble. The solid particles are then recovered from the slurry, as by filtering. In the second slurry washing step, the particles are slurried with a solvent for the non-polar impurities, in which solvent the particles are insoluble or, at most, only slightly soluble. The solid particles are then recovered from the slurry, as by filtering. If desired, one or both of the slurry washing steps may be repeated to remove additional impurities. However, preferably each washing is performed only once, to minimize loss of solids. For purposes of this application, particles of protected nucleoside will be considered to be slightly soluble in a solvent when less than about 10 wt % of the total particles dissolve during a washing step, and preferably less than about 5 wt % dissolve, to minimize loss.
While not wishing to be bound to a particular theory of how this process works, it is believed that when the solvent wets and slightly dissolves the surface of the particles, impurities which may be tied up or attached in any manner thereto are released into solution.
Therefore, although it is desirable to minimize the loss of solids, it is believed that improved washing is obtained when at least about 0.1 wt % of the solid particles of protected nucleoside dissolve in the solvent during a washing step, preferably at least about
Gaita Romulus
Koljack Mathias P.
Swedo Raymond J.
Wu Charles
Yates Stephen Frederick
Allied-Signal Inc.
Szuch Colleen
Wilson James O.
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