Methods of preparing and purifying 9-nitro-20-camptothecin

Details Methods of preparing and purifying 9-nitro-20-camptothecin Methods of preparing and purifying 9-nitro-20-camptothecin

2000-06-09

2004-01-27

Kifle, Bruck (Department: 1624)

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

Organic compounds

Heterocyclic carbon compounds containing a hetero ring...

Reissue Patent

active

RE038408

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to method of preparing and purifying 9-nitro-20-camptothecin (9NC).
2. Description of Related Art
9-nitro-20(S)-camptothecin has shown great promise in treating certain types of cancer. The water-insoluble 9-nitro-20(S)-camptothecin has been studied both in vitro and in vivo and 9-nitro-20(S)-camptothecin is in clinical trials for certain types of cancer.
Japanese Kokai Patent Application No. 59-51288 provides one method of making 9-nitro-camptothecin by treating camptothecin with a slight excess of concentrated nitric acid in concentrated sulfuric acid. However, when following this procedure, a yield of about 3% to about 7% of the 9NC product which is capable for medicinal use is obtained. Further, this procedure results in unwanted byproducts such as the inactive isomer, 12-nitrocamptothecin (12NC), in a ratio of about 1 to 3 of 9NC to 12NC. The following scheme shows the reaction from the use of nitric acid and sulfuric acid.
The unwanted 12NC, unfortunately, is the major product and accounts for about 60% of the yield. Because of the low yield of the 9NC and the numerous byproducts, the process of separating and purifying the 9NC is time consuming and costly and leads to a further decrease in the yield of 9NC. Accordingly, there is a need for a process that leads to a higher yield of 9NC and preferably without the numerous byproducts that have accompanied previous methods of making 9NC.
SUMMARY OF THE INVENTION
A feature of the present invention is to provide a method for the preparation of 9-nitrocamptothecin which preferably provides an increase in the yield of 9-nitrocamptothecin.
An additional feature of the present invention is to provide an improved purification procedure for purifying 9-nitrocamptothecin.
Additional features and advantages of the present invention will be set forth in part in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. The objectives and other advantages of the present invention will be realized and attained by means of the elements and combinations particularly pointed out in the written description and appended claims.
To achieve these and other advantages and in accordance with the purpose of the present invention, as embodied and broadly described herein, the present invention relates to a method for the preparation of 9-nitrocamptothecin. The method includes the steps of reacting 20-camptothecin with at least one inorganic nitrate salt and an acid which is effective in catalyzing the formation of a nitronium ion. The reaction occurs at a temperature and for a time sufficient to form the 9-nitrocamptothecin.
The present invention also relates to a method of purifying the 9-nitrocamptothecin by column chromatography which preferably uses an eluant comprising tetrahydrofuran and methylene chloride.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the present invention, as claimed.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
In general, the method for making 9-nitrocamptothecin involves reacting 20-camptothecin with at least one inorganic nitrate salt and an acid effective in catalyzing the formation of a nitronium ion from the nitrate salt. The reaction occurs at a temperature and for a time sufficient to form the 9-nitrocamptothecin.
With respect to the starting materials, the camptothecin can be any 20-camptothecin. Preferably, the 20-camptothecin is a racemic 20-camptothecin (i.e., 20(R, S)-camptothecin) or 20(S)-camptothecin. More preferably, the 20-camptothecin is 20(S)-camptothecin. The camptothecin is commercially available from such sources as Jingtao Science and Technology Development Co., Beijing, China. While any purity of camptothecin can be used, preferably, the 20-camptothecin has a purity of from about 85% to about 99%, more preferably at least about 92%. The amount of 20-camptothecin reacted with at least inorganic nitrate salt and an acid effective in catalyzing the formation of a nitronium ion can be any amount, as long sufficient amounts of the remaining ingredients are present.
The inorganic nitrate salt can be any salt which is capable of forming a nitronium ion which ultimately results in the nitro substituent attached at the 9-position of the camptothecin. Examples of inorganic salts include, but are not limited to, KNO
3
; NH
4
NO
3
; LiNO
3
; AgNO
3
; TiNO
3
; BiONO
3
; Cu(NO
3
)
2
2.5 H
2
O; Hg(NO
3
)
2
H
2
O; Ca(NO
3
)
2
4.H
2
O; Ba(NO
3
)
2
; Zn(NO
3
)
2
6.H
2
O; Mg(NO
3
)
2
6.H
2
O; Co(NO
3
)
2
6.H
2
O; Sr(NO
3
)
2
; Pb(NO
3
)
2
; Al(NO
3
)
3
9.H
2
O; Fe(NO
3
)
3
9.H
2
O; Cr(NO
3
)
3
9.H
2
O; and La(NO
3
)
3
6.H
2
O.
In the method of the present invention, at least one inorganic nitrate salt is used in the reaction. More than one inorganic nitrate salt can be used, for instance, two or three or more different inorganic nitrate salts can be used in the same reaction. Certain combinations of inorganic nitrate salts have lead to improved results with respect to the percent yield of 9-nitrocamptothecin and a more favorable ratio of the present of 9-nitrocamptothecin yield to the percent of 9-nitrocamptothecin yield as well as a higher total percent of nitration and/or a lower percent of byproducts resulting from the reaction.
Specific examples of combinations of inorganic nitrate salts include, but are not limited to, KNO
3
and Cu(NO
3
)
2
2.5.H
2
O; KNO
3
and TiNO
3
; KNO
3
and Hg(NO
3
)
2
H
2
O; KNO
3
and Ca(NO
3
)
2
4.H
2
O; KNO
3
and Ba(NO
3
)
2
; KNO
3
and Zn(NO
3
)
2
6.H
2
O; KNO
3
and Sr(NO
3
)
2
; KNO
3
and Pb(NO
3
)
2
; KNO
3
and Al(NO
3
)
3
9.H
2
O; KNO
3
and Fe(NO
3
)
3
9.H
2
O; LiNO
3
and Hg(NO
3
)
2
.H
2
O; LiNO
3
and Cu(NO
3
)
2
2.5.H
2
O; LiNO
3
and Co(NO
3
)
2
6.H
2
O; AgNO
3
and Cr(NO
3
)
3
9.H
2
O; Cu(NO
3
)
2
2.5.H
2
O and Fe(NO
3
)
3
9.H
2
O; Hg(NO
3
)
2
.H
2
O and Fe(NO
3
)
3
9.H
2
O; NH
4
NO
3
and Cu(NO
3
)
2
2.5.H
2
O; KNO
3
, TiNO
3
, and Cu(NO
3
)
2
2.5 H
2
O; KNO
3
, TiNO
3
, and Zn(NO
3
)
2
6.H
2
O; KNO
3
, TiNO
3
, and Pb(NO
3
)
2
; KNO
3
, Cu(NO
3
)
2
2.5.H
2
O, and Fe(NO
3
)
3
9.H
2
O; KNO
3
, LiNO
3
, Cu(NO
3
)
2
2.5.H
2
O, Hg(NO
3
)H
2
O, and Fe(NO
3
)
2
H
2
O; Zn(NO
3
)
2
6.H
2
O; KNO
3
, LiNO
3
, AgNO
3
, Cu(NO
3
)
2
2.5.H
2
O, Hg(NO
3
)
2
H
2
O, and Fe(NO
3
)
3
9.H
2
O; KNO
3
, LiNO
3
, Zn(NO
3
)
2
6.H
2
O. Cu(NO
3
)
2
2.5 H
2
O, Hg(NO
3
)
2
H
2
O, and Fe(NO
3
)
3
9.H
2
O; and KNO
3
, Zn(NO
3
)
2
6.H
2
O, Cu(NO
3
)
2
2.5.H
2
O, Hg(NO
3
)
2
H
2
O, and Fe(NO
3
)
3
9.H
2
O.
The amount of inorganic nitrate salt present in the reaction (as compared with the amount of camptothecin used in the reaction) is in excess of the amount of camptothecin used and more preferably is from about 2 times to about 3 times by mole more than the 2-camptothecin present. The inorganic nitrate salts are commercially available from such sources as Aldrich Chemical Co., Milwaukee, Wis.
With respect to the acid used in the method of the present application, the acid is effective in catalyzing the formation of a nitronium ion. Examples of acids include, but are not limited to, concentrated sulfuric acid, trifluoroacetic acid, or trifluoroacetic acid anhydride. By the use of the term “concentrated,” the sulfuric acid should have a concentration of at least about 95% and more preferably from about 96% to about 98%. The amount of acid used in the reaction should be in the range of 50 ml to 120 ml/gram of 20-CPT, more preferably, 100 ml/gram of 20-CPT.
In conducting the method of the present application, generally, the starting materials can be added to a reaction vessel in any sort of order and mixed together until the formation of the 9-nitrocamptothecin. While the order that the starting materials are added to the reaction vessel is not critical, it is preferred that the acid is first added to the reaction vessel which is equipped with a magnetic

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