Processes for producing P1,P4-diuridine-5′-tetraphosphate

Details Processes for producing P1,P4-diuridine-5′-tetraphosphate Processes for producing P1,P4-diuridine-5′-tetraphosphate

2002-04-12

2004-08-24

Wilson, James O. (Department: 1624)

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

Organic compounds

Carbohydrates or derivatives

C536S055300

Reexamination Certificate

active

06780988

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to stable crystals of P
1
,P
4
-di(uridine 5′-)tetraphosphate (U
2
P
4
) or a salt thereof which are useful as an active ingredient of an expectorant or a therapeutic agent for pneumonia; a process for producing the crystals; and a process for efficiently producing U
2
P
4
or a salt thereof.
BACKGROUND ART
A tetrasodium salt of P
1
,P
4
-di(uridine 5′-)tetraphosphate (U
2
P
4
.4Na) represented by the following formula (I):
has an expectoration-inducing action and is a compound which is expected to be developed as an expectorant or a therapeutic agent for pneumonia (e.g., U.S. Pat. Nos. 5,789,391, 5,763,447, and 5,635,160).
Until now, U
2
P
4
has not been obtained in crystal form, and has been prepared only in the form of a lyophilized product (see Example 1 of WO 99/05155). Typical U
2
P
4
produced by conventional method has a purity as low as 90%, and contains by-products. Examples of by-products include nucleoside 5′-(poly)phosphates such as uridine 5′-tetraphosphate (UP
4
), uridine 5′-triphosphate (UTP), uridine 5′-diphosphate (UDP), and uridine 5′-monophosphate (UMP); and dinucleoside polyphosphates such as P
1
,P
4
-di(uridine 5′-)triphosphate (U
2
P
3
) and P
1
,P
4
-di(uridine 5′-)diphosphate (U
2
P
2
). Particularly, it is difficult to separate nucleoside 5′-(poly)phosphates such as UTP from U
2
P
4
, and highly purified U
2
P
4
has been produced only with great difficulty through a conventional purification method; i.e., ion-exchange chromatography (WO 99/05155,
Biochimica et Biophysica Acta,
438, (1976) 304-309).
The above purified and lyophilized product has disadvantages such as high hygroscopicity. Therefore, preparation of a pharmaceutical from U
2
P
4
must be carried out in a special apparatus in which moisture is well controlled. Even after preparation of a pharmaceutical, the product must be wrapped tightly. In addition, since the pharmaceutical has a very short available period due to poor stability of the lyophilized preparation, obtaining highly purified and stable U
2
P
4
crystals has been desired.
U
2
P
4
is synthesized from uridine 5′-monophosphate (UMP) by use of an activating agent such as diphenyl phosphorochloridate (DPC) and a phosphorylating agent such as a pyrophosphate (PPi). However, a conventional process provides a low synthesis yield; i.e., as low as approximately 10 wt. % (Example 4B of WO 99/05155), and can never serve as a practical process. Accordingly, development of a process for producing U
2
P
4
at high yield and on a large scale has also been desired.
In view of the foregoing, an object of the present invention is to provide stable crystals of U
2
P
4
or a salt thereof. Another object of the invention is to provide a process for producing the crystals. Still another object of the invention is to provide a process for efficiently producing U
2
P
4
on a large scale.
DISCLOSURE OF THE INVENTION
The present inventors have conducted earnest studies on a method for purifying U
2
P
4
and a process for synthesizing U
2
P
4
from UMP. The inventors have found that U
2
P
4
purified through anion exchange chromatography and chromatography using activated charcoal (activated-charcoal chromatography) can be easily crystallized and that use of specific reaction conditions has the effect of drastically increasing the yield of U
2
P
4
in the synthesis of U
2
P
4
or a salt thereof from UMP serving as a starting material and by use of DPC and PPi. The present invention has been achieved on the basis of these findings.
Accordingly, the present invention provides crystals of P
1
,P
4
-di(uridine 5′-)tetraphosphate or a salt thereof.
The present invention also provides a process for producing crystals of P
1
,P
4
-di(uridine 5′-)tetraphosphate or a salt thereof, which process comprises purifying crude P
1
,P
4
-di(uridine 5′-)tetraphosphate or a salt thereof through anion exchange chromatography and activated-charcoal chromatography and adding a hydrophilic organic solvent to a solution of purified P
1
,P
4
-di(uridine 5′-)tetraphosphate or a salt thereof, to thereby precipitate crystals.
The present invention also provides a process for producing P
1
,P
4
-di(uridine 5′-) tetraphosphate or a salt thereof from uridine 5′-monophosphate (UMP) serving as a starting material and by use of diphenyl phosphorochloridate (DPC) and a pyrophosphate (PPi), which process comprises at least one of the following treatment steps:
(a) adding UMP diphenylphosphate (UMP-DPP) in divided portions during a step of reaction of UMP-DPP with a PPi-organic alkali salt;
(b) carrying out a step of reaction of UMP-DPP with a PPi-organic alkali salt in the presence of a base; and
(c) further treating the synthesized U
2
P
4
with an alkali.


REFERENCES:
patent: 5763447 (1998-06-01), Jacobus et al.
patent: 6319908 (2001-11-01), Yerxa et al.
patent: 216881 (1999-09-01), None
patent: WO-97/29756 (1997-08-01), None
patent: WO-97/35591 (1997-10-01), None
patent: WO-99/05155 (1999-02-01), None
Grant, R.L. et al, “Grant and Hackh's Chemical Dictionary”, McGraw-Hill, New York, 1987, pp. 22-23.*
Pauling, Linus, General Chemistry, 2nd Ed.:, 1953, Freeman, San Francisco, P22 & 41.
Coste, H. et al., J. Biol. Chem., 262, 1987, 12096-12103.
Vallejo, C.G. et al, Biochimica et Biophysica Acta, 438, 1976, 304-309.

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