Di-(uridine 5′)-tetraphosphate and salts thereof

Details Di-(uridine 5′)-tetraphosphate and salts thereof Di-(uridine 5′)-tetraphosphate and salts thereof

2001-11-20

2003-04-15

Richter, Johann (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C514S051000

Reexamination Certificate

active

06548658

ABSTRACT:

TECHNICAL FIELD
This invention relates to methods for the production of therapeutic dinucleotides including novel salts thereof. More specifically, it relates to methods for synthesis of P
1
, P
4
-di(uridine 5′)-tetraphosphate, i.e., diuridine tetraphosphate (U
2
P
4
) which have advantages over prior art methods of manufacture.
BACKGROUND OF THE INVENTION
P
1
, P
4
-Di(uridine 5′)-tetraphosphate is a dinucleotide of the following structure:
wherein:
X is Li, Na, K, NH
4
or H, provided that all X groups are not H.
The free acid of P
1
, P
4
-di(uridine 5′)-tetraphosphate, where X is hydrogen, has been previously described as uridine 5′-(pentahydrogen tetraphosphate), P′″→5′-ester with uridine (CAS Registry Number: 59985-21-6; C. Vallejo et al.,
Biochimica et Biophysica Acta
438, 305 (1976) and H. Coste et al., J. Biol. Chem. 262, 12096 (1987)).
Different methods have been described for the synthesis of purine dinucleotides such as diadenosine tetraphosphate (A
2
P
4
) (E. Rappaport et al,
Proc. Natl. Acad. Sci,
78, 838, (1981); A. Guranowski et al,
Biochemistry,
27, 2959, (1988); C. Lobaton et al, Eur. J. Biochem., 50, 495, 1975; K. Ng and L. Orgel,
Nucl. Acid Res.,
15, 3573, (1987)). However, this has not been true for U
2
P
4
which is a pyrimidine nucleotide. Although purine nucleotides and pyrimidine nucleotides appear to be analogous, the methods used for purine nucleotide synthesis do not necessarily work for pyrimidines such as uridine.
Diuridine tetraphosphate has been shown to have beneficial properties in the treatment of various diseases, such as chronic obstructive pulmonary disease (COPD). For example, they have been demonstrated to facilitate the clearance of mucous secretions from the lungs of a subject such as a mammal including humans in need of treatment for various reasons, including cystic fibrosis, chronic bronchitis, asthma, bronchiectasis, post-operative mucous retention, pneumonia, primary ciliary dyskinesia (M. J. Stutts, III, et al, U.S. Pat. No. 5,635,160; PCT International Publication WO 96/40059) and the prevention and treatment of pneumonia in immobilized patients (K. M. Jacobus and H. J. Leighton, U.S. Pat. No. 5,763,447). Further therapeutic uses include treatment of sinusitis (PCT International Publication WO 98/03177), otitis media (PCT International Publication WO 97/29756), dry eye, retinal detachment, nasolacrimal duct obstruction, the treatment of female infertility and irritation due to vaginal dryness via increased mucus secretions and hydration of the epithelial surface, and enhancing the performance of athletes.
U
2
P
4
also has utility as a veterinary product in mammals such as, but not limited to, dogs, cats and horses.
Prior art methodology describes only one protocol for the production of diuridine tetraphosphate. This method is very time consuming, lasting over five days and producing only small amounts of diuridine tetraphosphate (C. Vallejo et al.,
Biochimica et Biophysica Acta
438, 305 (1976), Sillero et al.,
Eur J Biochem
76, 332 (1972)). According to this technique, diuridine tetraphosphate was synthesized through a reaction of uridine 5′-monophosphomorpholidate (0.54 mmol) with the triethylamine salt of pyrophosphoric acid (0.35 mmol) in a medium of anhydrous pyridine (10 ml). After 5 days at 30° C., pyridine was removed from the reaction mixture by evaporation, and the residue resuspended in glass-distilled water (8 mL), the suspension applied to a DEAE-cellulose column (37.5×2.6 cm) and fractionated with 3.2 L of a linear gradient (0.06-0.25 M) of ammonium bicarbonate, pH 8.6. The peak eluting between 0.17-0.19 M ammonium bicarbonate was partially characterized as U
2
P
4
by the following criteria: insensitivity to alkaline phosphatase, phosphorus to base ratio and analysis of the products of hydrolysis (UTP+UMP), after treatment with phosphodiesterase I, by electrophoresis in citrate buffer, pH 5.0. No yield or spectroscopic data were given. Thus, the prior art procedure for the synthesis of diuridine tetraphosphate is lengthy and produced only small amounts of only partially characterized diuridine tetraphosphate. The present invention focuses on methods to produce this medically useful compound which may be more efficiently and conveniently carried out, and which may be applied to the large-scale production of diuridine tetraphosphate and salts thereof.
SUMMARY OF THE INVENTION
The present invention provides new methods for the synthesis of the therapeutic dinucleotide, P
1
, P
4
-di(uridine 5′)-tetraphosphate (Formula I), and demonstrates applicability to the production of large quantities. The methods of the present invention substantially reduce the time required to synthesize diuridine tetraphosphate, preferably to three days or less. The ammonium, sodium, lithium, and potassium salts of P
1
, P
4
-di(uridine 5′)-tetraphosphate prepared by these methods are stable, soluble, nontoxic, and easy to handle during manufacture. The tetrasodium, tetraammonium, tetralithium and tetrapotassium salts of P
1
, P
4
-di(uridine 5′)-tetraphosphate (Formula I) prepared by these methods are highly pure and stable.
wherein:
X is Na, NH
4
, Li, K, or H, provided that all X groups are not H.
The method of synthesizing compounds of Formula I, and pharmaceutically acceptable salts thereof, is carried out generally by the following steps: 1) dissolving uridine or uridine nucleotide compounds of Formulas IIa-d in a polar, aprotic organic solvent and a hydrophobic amine; 2) phosphorylating with a phosphorylating agent of one of the Formulas IVa-b to yield a compound of Formula I, or activating a phosphate group of the uridine nucleotide compound with an activating agent of one of the Formulas IIIa-c and reacting with a suitable compound of Formula II b-d to yield a compound of Formula I; and 3) purifying by ion exchange chromatography.
Another aspect of the present invention are methods of treating various disease states, including, but not limited to: chronic obstructive pulmonary diseases, sinusitis, otitis media, nasolacrimal duct obstruction, dry eye disease, retinal detachment, pneumonia, and female infertility or irritation caused by vaginal dryness.
Another aspect of the present invention is a pharmaceutical composition comprising a compound of Formula I together with a pharmaceutically acceptable carrier.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides new methods for the synthesis of the therapeutic dinucleotide, P
1
, P
4
-di(uridine 5′)-tetraphosphate, and demonstrates applicability to the production of large quantities. The methods of the present invention substantially reduce the time period required to synthesize P
1
, P
4
-di(uridine 5′)-tetraphosphate, preferably to three days or less. The ammonium, potassium, lithium and sodium salts of P
1
, P
4
-di(uridine 5′)-tetraphosphate (Formula I) prepared by these methods are stable, soluble, nontoxic, and easy to handle during manufacture.
The present invention further provides compounds of Formula I:
wherein:
X is Na, NH
4
, Li, K, or H, provided that all X groups are not H.
The sodium, ammonium, lithium and potassium salts of P
1
, P
4
-di(uridine 5′) -tetraphosphate have many advantages, for example, they provide good long-term stability profiles compared to those of divalent cations (e.g. Ca
2+
, Mg
2+
, Mn
2+
) which catalyze hydrolysis of phosphate esters.
These inorganic sodium, ammonium, lithium, and potassium salts impart excellent water solubility compared to hydrophobic amine salts such as tri- and tetrabutylammonium, and similar salts. High water solubility is an important advantage for flexibility in pharmaceutical formulations of varying concentration.
The tetrammonium, tetrasodium, tetralithium and tetrapotassium salts of P
1
, P
4
-di(uridine 5′)-tetraphosphate are additionally advantageous in that they are readily purified by aqueous ion chromatography in which no organic solvents are used. They ha

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