Abstract:
An Estracyt compound having a carcinostatic bound thereto is obtained by reacting an Estracyt compound with a carcinostatic having one or more radicals selected from among COOH, Cl, NH 2  and OH, either directly or after reaction with an amine to replace one or both Cl groups in the nitrogen mustard portion in the Estracyt compound with a NH 2  group, in the presence or absence of a catalyst. The resulting compound is more effective in cancer control than the Estracyt compound associated substance.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a process for preparing Estracyt compounds having a carcinostatic bound thereto, useful in the treatment of prostatic cancer. 
     2. Description of the Prior Art 
     Studies on hormone-dependent cancers have been considerable adavnce in recent years and one approach that is considered effective is transporting a high concentration of a hormone-based carcinostatic to a specific site in the cancer tissues and letting the carcinostatic exhibit its cancer control activity at that site. Estracyt® is the trademark for estradiol-3N-bis(2-chloroethyl)-carbamate-17β-phosphate that Ab-Leo A.G., Sweden developed in 1966 for achieving this purpose. Estracyt® has the following formula (I): ##STR1## 
     Estracyt® is a substance wherein estradiol-17β-phosphate of formula (II) has a carbamate bonding of nitrogen mustard of formula (III) at 3-position: ##STR2## 
     Two derivatives having the same activity as Estracyt® are estramustine of formula (IV) and estromustine of formula (V): ##STR3## 
     It has been reported that Estracyt®, estramustine and estromustine (hereunder collectively referred to as &#34;Estracyt compounds&#34;), when administered into the human body, are specifically accumulated in prostatic organs (e.g. dorsal prostate, DP; dorsolateral prostate, DLP; and seminal vesicle, SV) (see, for example, H. Yamanaka, K. Kitaura, K. Imai, H. Yuasa, K. Nakai, Y. Matsumura, H. Uehara and K. Shiba, &#34;In Vivo Studies of  3  H-estramustine in Castrated Male Rat&#34;, Acta Urol. Jap., 27, 243-250, 1981). It has also been reported that these Estracyt compounds are very effective in controlling progressive prostatic cancer. 
     Estracyt compounds may function as estrogen-cancer control agent complexes but their cancer control activities are not completely satisfactory. 
     SUMMARY OF THE INVENTION 
     The present inventors, therefore, made attempts at realizing more effective cancer treatment by combining a carcinostatic with one of the Estracyt compounds that are specifically accumulated in certain organs. As a result, the inventors have found a compound having bound thereto an agent that is more effective in cancer control than the Estracyt compound associated substance. The present invention has been accomplished on the basis of this finding. 
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The problem stated above can be solved by a compound having an Estracyt compound bound to a carcinostatic having one or more radicals selected from among COOH, Cl, NH 2  and OH. 
     The compound in accordance with the present invention is hereunder described with particular reference to estramustine as the Estracyt compound to be bound with a carcinostatic. 
     In order to bind a carcinostatic to estramustine of the formula ##STR4## one or both Cl groups in the nitrogen mustard portion may be bound to the carcinostatic either directly or indirectly. In the indirect fashion, the nitrogen mustard may be bound to NH 3  or H 2  N--(CH 2 ) n  --NH 2  so as to convert one or both Cl groups to NH 2  groups, as shown below. ##STR5## The introduction of the NH 2  group is typically done at 20°-100° C., preferably 40°-70° C. The compound having one or both Cl groups in the Estracyt compound replaced by NH 2  groups is hereunder sometimes abbreviated as Est--NH 2 . The object compound of the present invention can be obtained by reacting the Est--NH 2  with a known carcinostatic having COOH, Cl, NH 2  or OH group. The specific methods for binding these radicals in carcinostatics with Est--NH 2  are carried out in the presence of a catalyst or in the absent thereof indicated below. ##STR6## 
     According to the present invention, the Cl group in the Estracyt compound may be directly bound to the NH 2  group in the carcinostatic, but the direct method does not achieve a very high binding efficiency. The more preferred method is the indirect one wherein the Estracyt compound is bound with a carcinostatic after one or both Cl groups in the Estracyt compound are converted to NH 2  groups. 
     Illustrative carcinostatics that may be bound to the Estracyt compound in accordance with the present invention are given below, with the radicals present in the carcinostatics being indicated within parentheses: chlorombucil (Cl, COOH), cyclophosphamide (Cl), mannomustine (OH, Cl), melphalan (Cl, COOH, NH 2 ), carbazilgunone (NH 2 ), cytarabine (NH 2 , OH), methotrexate (NH 2 , COOH), sodium aminopterin (NH 2 , COOH), sarcomycin (COOH), dactinomycin (NH 2 ), mitomycin C (NH 2 ), guanylhyclrazone (NH 2 ) and doxorubicin hydrochloride (NH 2 ). Any other carcinostatics having COOH, Cl, NH 2  or OH group may be bound to the Estracyt compounds. 
    
    
     The advantages of the present invention are hereunder described in greater detail by reference to the following Examples. 
     EXAMPLE 1 
     Estracyt® (0.5 g) was dissolved in a commercial aqueous NH 3  (5 ml) and phosphate buffer solution (PBS, 20 ml, pH 7.2) and the solution was heated at 50° C. for 2 days. By this treatment, the Cl portion in Estracyt® was converted to NH 2 . To the resulting Estracyt®-NH 2 , a mixture of PBS (5 ml, pH 7.2) and 0.1% glutaraldehyde solution (0.5 ml) containing 50 mg of doxorubicin hydrochloride of the formula indicated below was added: ##STR7## By subsequent treatment at 40° C. for 3 days, Estracyt® was chemically bound to the NH 2  group in the doxorubicin hydrochloride. 
     REFERENCE EXAMPLE 1 
     The Estracyt®-doxorubicin hydrochloride compound prepared in Example 1 and which contained 30 μg of Estracyt® was injected into male Wistar rats (average weight: 400 g) through femoral blood vessels. On week later, the rats were killed and their prostatic organs (DP, DLP and SV) were removed. When 30 μg of Estracyt® was injected alone, the contraction of the prostatic organs was negligible. On the other hand, the degree of contraction of the prostatic organs extracted from the treated group was about 40% greater than in the control group. Optical microscopic observations showed marked necrosis in the SV, DLP and VP stained with hematoxylineosin, but other organs (e.g., liver, heart, kidney, lung, spleen, pancrease and testicle) showed little sign of necrosis. 
     EXAMPLE 2 
     Estramustine (1 g) was treated with a mixture of ethylenediamine (7 ml) and ethyl alcohol (20 ml) at 50° C. for 3 days so as to convert the Cl portion in the estramustine to --NHCH 2  CH 2  NH 2 . To the resulting estramustine--NHCH 2  CH 2  NH 2  compound, 10 ml of ethyl alcohol containing mitomycin C (80 mg) and 30 mg of glutaraldehyde were added, and the mixture was heated at 45° C. for 2 days. By this treatment, the estramustine was bound chemically with the NH 2  group in mitomycin C. 
     REFERENCE EXAMPLE 2 
     The estramustine-mitomicin C compound prepared in Example 2 and which contained 40 μg of estramustine was injected into male Wistar rats through femoral blood vessels. One week later, the rats were slaughtered and their prostatic organs were removed as in Reference Example 1. 
     Estramustine, when administered independently, caused little contraction of the prostatic organs. However, the degree of contraction of the prostatic organs extracted from the treated group was about 50% greater than in the control group. Other results were similar to those obtained in Reference Example 1. 
     EXAMPLE 3 
     Estramustine (0.5 g) was treated with a mixture of octamethylenediamine (3 g) and benzene (20 ml) at 60° C. for one day so as to convert the Cl portion in the estromustine to --NH(CH 2 ) 8  NH 2 . To the resulting estromustine--NH(CH 2 ) 8  NH 2  30 mg of mitomycin C and 10 ml of glutaraldehyde were added and the mixture was heated at 40° C. for 2 days. By this treatment, the estromustine was chemically bound to the NH 2  group in mitomycin C. 
     REFERENCE EXAMPLE 3 
     The estromustine-mitomycin C compound prepared in Example 3 and which contained 40 μg of estromustine was injected into male Wistar rats through femoral blood vessels. One week later, the rats were slaughtered and their prostatic organs were removed. The experimental results were substantially the same as in Reference Example 1. 
     EXAMPLE 4 
     An Estracyt®-chlorambucil compound was prepared as in Example 1 except that doxorubicin hydrochloride was replaced by chlorambucil and, glutaraldehyde by 1-ethyl-3-(3-dimethylaminopropyl)-carbadiimide. 
     REFERENCE EXAMPLE 4 
     The Estracyt®-chlorambucil compound prepared in Example 4 and which contained 30 μg of Estracyt® was injected into male Wistar rats through femoral blood vessels. One week later, the rats were killed and their prostatic organs were removed. The extracted organs had suffered about 20% contraction but the degree of their necrosis was substantially the same as in Reference Example 1.